Three-dimensional polymer networks with channels situated therein

ABSTRACT

The disclosure provides three-dimensional crosslinked polymer networks comprising one or more channels extending from the surface and/or near the surface of the network into the interior of the network, arrays comprising the networks, processes for making the networks, and uses of the networks and arrays.

1. CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. § 119 to Europeanapplication no. 15201355, filed Dec. 18, 2015 and to U.S. applicationSer. No. 15/008,728, filed Jan. 28, 2016, the contents of each of whichare incorporated herein in their entireties by reference thereto.

2. BACKGROUND

U.S. Publication No. 2008/0293592 describes a method for covalentlyimmobilizing probe-biomolecules on organic surfaces by means ofphotoreactive crosslinking agents. The method has in practice proven tobe advantageous particularly because it permits an immobilization ofprobe biomolecules on unreactive surfaces, such as silanized glasssupports and substrates made of standard commercial plastics. A polymeris used in the method described in US 2008/0293592 to form a type ofthree-dimensional network onto which the probe biomolecules can bebonded, either at the network's surface or in the inside of the network.Compared to an organic surface on which the probe biomolecules are onlyimmobilized in two-dimensional form, the three-dimensionalimmobilization of the biomolecules in the polymer and/or copolymernetwork permits a higher density of the probe biomolecules on theorganic surface. This increases the amount of analyte which can bebonded per surface unit of the organic surface. Use of the surface asbiological sensor thus gives rise to a higher measurement accuracy and ahigh measurement dynamic.

However, a disadvantage of the methods and polymer networks described inU.S. 2008/0293592 is that analyte molecules or analyte components whichbind to probe biomolecules arranged on or close to the surface of thepolymer network can block the network. Further analyte molecules oranalyte constituents can then no longer bind as well to as yet unboundprobe biomolecules which are arranged at a greater distance from thesurface of the network in its interior.

Thus, there is a need for improved polymer networks.

3. SUMMARY

This disclosure provides three-dimensional polymer networks comprising acrosslinked polymer and one or more channels that extend from a surfaceand/or near a surface of the network into the network's interior. Thenetworks are suitably covalently attached to a surface. One or moreprobes, such as a biomolecule, can be immobilized on the surface of thenetwork and throughout the interior of the network, providing a sensorfor detecting the presence of and/or measuring the amount of an analytein a sample. For example, nucleic acid probes can be used to detectcomplementary nucleic acids present in a sample and antibody probes canbe used to detect antigens present in a sample. The networks of thedisclosure allow for faster hybridization of a given amount of analytethan networks lacking channels because the channels can effectivelyincrease the surface area of the network, exposing more probes to thesample in a given amount of time. Additionally, the networks of thedisclosure can bind more analyte than the same volume of a channel-freenetwork because the channels decrease or eliminate the problem wherebyanalyte or other components of a sample bound to probes at or near thesurface of the network block access to probes located in the interior ofthe network. Another advantage of the networks of the disclosure is thatthe high amount of analyte loading made possible by the channels allowsfor a more sensitive detection of analyte than may be possible with achannel-free network, i.e., the signal to noise ratio can be improvedcompared to channel-free networks because a given amount of analyte canbe concentrated in a smaller network volume. Yet another advantage ofthe networks of the disclosure is that the high analyte loading madepossible by the channels allows for quantification of a wider range ofanalyte concentrations compared to channel-free networks.

This disclosure also provides arrays comprising a plurality of thethree-dimensional networks of the disclosure and a substrate. Arrays ofthe disclosure can be used to detect and/or measure one or more analytesin one or more samples simultaneously. The arrays of the disclosure canbe washed and reused, providing a significant cost advantage over singleuse arrays. Another advantage of the arrays of the disclosure is thatthey can be manufactured in a simple manner because all of thecomponents needed to make an individual network can be applied as asingle mixture onto a surface of the substrate during the manufacturingprocess.

This disclosure also provide processes for making the three-dimensionalnetworks and arrays of the disclosure. The three-dimensional networks ofthe disclosure can be made by crosslinking a polymer in the presence ofsalt crystals, preferably needle-shaped salt crystals, and subsequentlydissolving the salt crystals to leave behind channels in the crosslinkedpolymer network.

This disclosure also provides processes for using the three-dimensionalnetworks and arrays of the disclosure to detect and/or measure ananalyte in a sample, preferably a liquid sample.

4. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a diagrammatic representation of a mixture which has aprobe biomolecule (1) and a polymer (3) comprising two photoreactivegroups (4) per molecule dissolved in an aqueous salt solution.

FIG. 2 shows a cross-section through a drop of the mixture (5) shown inFIG. 1 having a surface (10) located at a spot (7) of an organic surface(2) which is situated on a heated holder (6).

FIG. 3 shows a cross-section through the arrangement shown in FIG. 2after the mixture has been heated and needle-shaped salt crystals (8)extending from crystallization germs (9) have been formed in the saltsolution.

FIG. 4 shows a cross-section through the arrangement shown in FIG. 3after the mixture has been dried and irradiated with optical radiation(11) to form a polymer network (15) having a surface (16).

FIG. 5 shows a diagrammatic representation of the mixture of FIG. 1following irradiation with optical radiation.

FIG. 6 shows a cross-section through the arrangement shown in FIG. 4after dissolving the salt crystals in a solvent (12), forming channels(13).

FIG. 7 shows a cross-section through the arrangement shown in FIG. 2after the mixture has been cooled on a chilled holder (14) andneedle-shaped salt crystals have been formed in the salt solution.

FIG. 8 shows a cross-section through the arrangement shown in FIG. 7after the mixture has been dried and irradiated with opticalirradiation.

FIG. 9 shows a cross-section through the arrangement shown in FIG. 8following dissolution of the salt crystals with a solvent.

FIG. 10 shows a reaction pathway for the formation ofp(Dimethylacrylamide co methacryloyloxybenzophenone co Sodium4-vinylbenzenesulfonate).

FIG. 11 shows a perspective view of a biochip (17) having an organicsurface (2) on which polymer networks (15) are located at spots (7)arranged as a matrix of rows and columns.

FIG. 12 shows a top view of a biochip (17) as shown in FIG. 11, whereeach polymer network (15) has a diameter (D), and where the rows andcolumns are separated by a distance Y and a distance X, respectively,measured from the center points of the polymer networks (15).

FIG. 13 shows a biosensor (17′) comprising a flexible substrate band(18) having an organic surface (2) on which polymer networks (15) havinga diameter (D) are located at spots (7) separated by distance X measuredfrom the center points of the polymer networks.

FIG. 14 shows a table showing the mean of the measured values andstandard error of the mean, for different spots in Example 2.

FIG. 15 is a graphic representation of the mean of the measured valuesobtained by the array of Section 7.2.1.

FIG. 16 is a graphic representation of the mean of the measured valuesobtained by an array of Section 7.2.2.

5. DETAILED DESCRIPTION 5.1. Three-Dimensional Polymer Networks

The three-dimensional networks of the disclosure comprise a crosslinkedpolymer, e.g., a polymer according to Rendl et al., 2011, Langmuir27:6116-6123 or US 2008/0293592, the contents of which are incorporatedby reference in their entireties herein. The three-dimensional networksof the disclosure further comprise one or more channels and canoptionally further comprise one or more probes immobilized on thenetwork.

Polymers that can be used to make the networks are described in Section5.1.1. Cross-linkers than can be used to make the networks are describedin Section 5.1.2. Features of the one or more channels are described inSection 5.1.3. Probes that can be immobilized on the networks aredescribed in Section 5.1.4.

5.1.1. Polymers

The three-dimensional networks of the disclosure can comprise acrosslinked homopolymer, copolymer, mixtures of homopolymers, mixturesof copolymers, or mixtures of one or more homopolymers and one or morecopolymers. The term “polymer” as used herein includes both homopolymersand/or copolymers. The term “copolymer” as used herein includes polymerspolymerized from two or more types of monomers (e.g., bipolymers,terpolymers, quaterpolymers, etc.). Copolymers include alternatingcopolymers, periodic copolymers, statistical copolymers, randomcopolymers, block copolymers, linear copolymers and branched copolymers.The three-dimensional networks of the disclosure can comprise anycombination of the foregoing types of polymers. Reagents and methods formaking such polymers are known in the art (see, e.g., Ravve, A.,Principles of Polymer Chemistry, Springer Science+Business Media, 1995;Cowie, J. M. G., Polymers: Chemistry & Physics of Modern Materials,2^(nd) Edition, Chapman & Hall, 1991; Chanda, M., Introduction toPolymer Science and Chemistry: A Problem-Solving Approach, 2^(nd)Edition, CRC Press, 2013; Nicholson, J. W., The Chemistry of Polymers,4^(th) Edition, RSC Publishing, 2012; the contents of each of which areherein incorporated by reference in their entirety).

Preferred polymers are hydrophilic and/or contain hydrophilic groups.The polymer can be water soluble. In an embodiment, the polymer is acopolymer that has been polymerized from two or more species of monomersselected to provide a desired level of water solubility. For example,water solubility of a copolymer can be controlled by varying the amountof a charged monomer, e.g., sodium 4-vinylsulfonate, used to make thecopolymer.

When crosslinked, water soluble polymers form water-swellable gels orhydrogels. Hydrogels absorb aqueous solutions through hydrogen bondingwith water molecules. The total absorbency and swelling capacity of ahydrogel can be controlled by the type and degree of cross-linkers usedto make the gel. Low crosslink density polymers generally have a higherabsorbent capacity and swell to a larger degree than high crosslinkdensity polymers, but the gel strength of high crosslink densitypolymers is firmer and can maintain network shape even under modestpressure.

A hydrogel's ability to absorb water is a factor of the ionicconcentration of the aqueous solution. In certain embodiments, ahydrogel of the disclosure can absorb up to 50 times its weight (from 5to 50 times its own volume) in deionized, distilled water and up to 30times its weight (from 4 to 30 times its own volume) in saline. Thereduced absorbency in saline is due to the presence of valence cations,which impede the polymer's ability to bond with the water molecule.

The three-dimensional network of the disclosure can comprise a copolymerthat has been polymerized from one, two, three, or more than threespecies of monomers, wherein one, two, three or more than three of thespecies of monomers have a polymerizable group independently selectedfrom an acrylate group (e.g., acrylate, methacrylate, methylmethacrylate, hydroxyethyl methacrylate, ethyl acrylate, 2-phenylacrylate), an acrylamide group (e.g., acrylamide, methacrylamide,dimethylacrylamide, ethylacrylamide), an itaconate group (e.g.,itaconate, 4-methyl itaconate, dimethyl itaconate) and a styrene group(e.g. styrene, 4-methyl styrene, 4-ethoxystyrene). Preferredpolymerizable groups are acrylate, methacrylate, ethacrylate, 2-phenylacrylate, acrylamide, methacrylamide, itaconate, and styrene. In someembodiments, one of the monomers used to make the copolymer is charged,e.g., sodium 4-vinylbenzenesulfonate.

The polymer used to make a network of the disclosure can comprise atleast one, at least two, or more than two cross-linker groups permolecule. A cross-linker group is a group that covalently bonds thepolymer molecules of the network to each other and, optionally, toprobes and/or a substrate. Copolymers that have been polymerized fromtwo or more monomers (e.g., monomers having a polymerizable groupindependently selected from those described in the preceding paragraph),at least one of which comprises a cross-linker, are suitable for makinga three-dimensional network of the disclosure. Exemplary cross-linkersare described in Section 5.1.2. A preferred monomer comprising across-linker is methacryloyloxybenzophenone (MABP) (see FIG. 10).

In a preferred embodiment, the copolymer is a bipolymer or a terpolymercomprising a cross-linker. In a particularly preferred embodiment, thecopolymer comprises p(Dimethylacrylamide co methacryloyloxybenzophenoneco Sodium 4-vinylbenzenesulfonate) (see FIG. 10).

5.1.2. Cross-Linkers

Crosslinking reagents (or cross-linkers) suitable for making thecrosslinks in the three-dimensional networks include those activated byultraviolet light (e.g., long wave UV light), visible light, and heat.Exemplary cross-linkers activated by UV light include benzophenone,thioxanthones (e.g., thioxanthen-9-one, 10-methylphenothiazine) andbenzoin ethers (e.g., benzoin methyl ether, benzoin ethyl ether).Exemplary cross-linkers activated by visible light include ethyl eosin,eosin Y, rose bengal, camphorquinone and erythrosin. Exemplarycross-linkers activated by heat include 4,4′ azobis(4-cyanopentanoic)acid, and 2,2-azobis[2-(2-imidazolin-2-yl) propane] dihydrochloride, andbenzoyl peroxide. Other cross-linkers known in the art, e.g., thosewhich are capable of forming radicals or other reactive groups uponbeing irradiated, may also be used.

5.1.3. Channels

The three-dimensional networks of the disclosure contain one or morechannels.

As used herein, a “channel” is an elongated passage in a network that(1) is substantially straight, and (2) in the hydrated state of thenetwork, has a minimum cross-section that is at least 500 nm and alength that is at least five times, and preferably at least ten times,the minimum cross-section of the passage. For example, the length of thechannel can be 5 to 15 times, 5 to 10 times, or 10 to 15 times theminimum cross-section of the channel. A channel that is “substantiallystraight” is one which extends from a point of nucleation in onedirection without changing direction more than 45 degrees in anydirection, i.e., the X, Y or Z direction.

The “hydrated state of the network” means that the network is atequilibrium with respect to water absorption, i.e., it absorbs inaqueous solution as much water as it emits.

Channels can allow access to the interior of the network. Althoughchannels can have a relatively large channel cross-section, the networkcan remain mechanically stable because the mesh size of the network canbe significantly smaller than the channel cross-section. The channelscan form a sort of highway, through which analytes can enter quicklyinto the interior of the network. The transport of the analytes can beeffected in the channel by diffusion and/or convection.

The channels can extend from a surface or near the surface of thenetwork into the interior of the network. For example, the one or morechannels can extend from a point that is less than 10 microns, less than9 microns, less than 8 microns, less than 7 microns, less than 6microns, less than 5 microns, less than 4 microns, less than 3 microns,less than 2 microns, less than 1 micron from the surface of the network,or extends into the interior from a point on the surface of the network.The network can contain a plurality of channels (e.g., 2 to 100, 2 to50, 2 to 25, 2 to 10, 10 to 50, 10 to 25, or 25 to 50), each of whichcan extend from a surface or near a surface of the network into theinterior of the network. In preferred embodiments, the network contains10, 20, 30, 40 or 50 channels, or a number of channels ranging betweenany two of the foregoing values (e.g., 10 to 50, 20 to 40, 30 to 50, 10to 20, 20 to 30, 30 to 40, or 40 to 50 channels). In a specificpreferred embodiment, the network contains 10 to 50 channels.

In some embodiments, the length of at least one channel is 10 to 100%,10% to 90%, 10% to 80%, 10% to 70%, 10% to 60%, 10% to 50%, 10% to 40%,10% to 30%, 10% to 15%, 10% to 20%, 10% to 25%, 10 to 30%, 15% to 20%,15% to 25%, 15% to 30%, 20 to 25%, or 25% to 30% of the largestdimension of the network. In preferred embodiments, the length of atleast one channel is approximately 10%, 20%, 30%, 40% or 50% of thelargest dimension of the network, and in some embodiments has a lengthranging between any pair of the foregoing embodiments (e.g., 10% to 50%,10% to 30%, 30% to 50%, 10% to 20%, 20% to 30%, 30% to 40%, or 40% to50% of the largest dimension of the network). In a specific preferredembodiment, the length is 10% to 50% of the largest dimension of thenetwork.

In some embodiments, the network comprises at least one channel having aminimum cross-section of at least 5 times, at least 10 times, at least15 times, or at least 20 times the mesh size (e.g., 5 to 10 times, 5 to15 times, 5 to 20 times, 5 to 25 times, 5 to 30 times, 10 to 15 times,10 to 20 times, 10 to 25 times, 10 to 30 times, 10 to 15 times, 10 to 20times, 10 to 25 times, 10 to 30 times, 15 to 20 times, 15 to 25 times,15 to 30 times, 20 to 25 times, 20 to 30 times, or 25 to 30 times thenetwork's mesh size). In preferred embodiments, the network comprises atleast one channel having a minimum cross-section of 10 to 30 times themesh size. This ensures a high stability of the polymer network, and canalso prevent network penetration and binding by undesirable largermolecules or components in a sample.

The network can have a mesh size (measured in the hydrated state of thenetwork) of, for example, 5 to 75 nm (e.g., 10 to 20 nm, 10 to 30 nm, 10to 40 nm, 10 to 50 nm, 20 to 30 nm, 20 to 40 nm, 20 to 50 nm, 30 to 40nm, 30 to 50 nm, or 40 to 50 nm).

The networks of the disclosure can comprise a plurality of channels(e.g., 2 to 100, 2 to 50, 2 to 25, 2 to 10, 10 to 50, 10 to 25, or 25 to50), and each channel can independently have one or more of the featuresdescribed in this section. In some embodiments, the majority of thechannels have one or more features described in this section. In aspecific preferred embodiment, the network contains 10 to 50 channelsthat each have one or more of the features described in this section.

Preferred three-dimensional networks contain a plurality of channelsthat converge at a point located within the network, and are arrangedsuch that, starting from the surface of the network towards theinterior, the lateral distance between the channels decreases. In someembodiments, a plurality of channels extend approximately radially awayfrom a point situated in the interior of the network. In someembodiments, the three-dimensional network contains multiple pluralitiesof channels (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 pluralities ofchannels), each plurality converging at a different point within thenetwork. In certain aspects, a plurality (or each plurality) areconnected at the point of their convergence.

The presence of a channel in a network can be verified using thefollowing procedure:

The network is brought into contact with an aqueous liquid at roomtemperature, for example, in a bowl. The liquid contains a plurality ofnanoparticles which are larger than the mesh size of the network andsmaller than the minimum cross-section of the channel. Thus,nanoparticles can enter the channel and spread along the channel.Without being bound by theory, it is believed that this can occur due tothe Brownian molecular motion and/or convection through the liquid inthe channel. Such nanoparticles are known as quantum dots. They can, forexample, have a diameter of about 10 nanometers.

An incubation period is selected so that the network in the liquid iscompletely hydrated, i.e., that the network on average takes the sameamount of water as it releases. The incubation period can be, forexample, one hour. The penetration of the nanoparticles in the channelcan be accelerated by setting in motion the network and/or the liquidduring the incubation, for example, by vibrating the network and/orliquid, preferably by means of ultrasonic waves.

After completion of the incubation, the liquid is separated from thenetwork, for example, by draining the liquid from the bowl or taking thenetwork out of the bowl.

Then, the hydrated network is frozen, for example, by means of liquidnitrogen. Thereafter, the frozen network can be cut with the aid of acryomicrotome along mutually parallel cutting planes into thin slices.The cutting planes are arranged transversely to the longitudinalextension of the channel and penetrate the channel. The cutting ispreferably carried out using a liquid nitrogen-cooled diamond blade. Thethickness of the slices can be, for example, about 100 nm or 200 nm.

With the aid of a microscope, the nanoparticles disposed in the disksobtained by cutting the frozen network are located. The nanoparticlescan be fluorescent and optically highlighted so that they can be betterdistinguished from the network, if necessary. The locating of thenanoparticles can be done using a suitable software with imageprocessing methods. To examine the disks, preferably a confocalmicroscope laser scanning microscope with fluorescence optics or anelectron microscope is used.

The geometry and/or position information of the nanoparticles obtainedin this manner may be, with the aid of a computer, used to make athree-dimensional geometric model of distribution of the nanoparticlesin the network. The model can then be used to determine whether thearrangement of the nanoparticles in the network comprises at least onesubstantially straight region whose cross-section is in no place smallerthan 500 nm and whose length corresponds at least to the five-fold ofits smallest cross-section. If this condition is fulfilled it isdetermined that the network comprises at least one channel.

Alternatively, the three-dimensional distribution of the nanoparticlescan be determined in the network by means of micro-3D X-ray computertomography.

5.1.4. Probes

A probe immobilized on the network of the disclosure can be abiomolecule or a molecule that binds a biomolecule, e.g., a partner of aspecifically interacting system of complementary binding partners(receptor/ligand). For example, probes can comprise nucleic acids andtheir derivatives (such as RNA, DNA, locked nucleic acids (LNA), andpeptide nucleic acids (PNA)), proteins, peptides, polypeptides and theirderivatives (such as glucosamine, antibodies, antibody fragments, andenzymes), lipids (e.g., phospholipids, fatty acids such as arachidonicacid, monoglycerides, diglycerides, and triglycerides), carbohydrates,enzyme inhibitors, enzyme substrates, antigens, and epitopes. Probes canalso comprise larger and composite structures such as liposomes,membranes and membrane fragments, cells, cell lysates, cell fragments,spores, and microorganisms.

A specifically interacting system of complementary bonding partners canbe based on, for example, the interaction of a nucleic acid with acomplementary nucleic acid, the interaction of a PNA with a nucleicacid, or the enzyme/substrate, receptor/ligand, lectin/sugar,antibody/antigen, avidin/biotin or streptavidin/biotin interaction.

Nucleic acid probes can be a DNA or an RNA, for example, anoligonucleotide or an aptamer, an LNA, PNA, or a DNA comprising amethacryl group at the 5′ end (5′ Acrydite™). Oligonucleotide probes canbe, for example, 12 to 30, 14 to 30, 14 to 25, 14 to 20, 15 to 30, 15 to25, 15 to 20, 16 to 30, 16 to 25, 16 to 20, 15 to 40, 15 to 45, 15 to50, 15 to 60, 20 to 55, 18 to 60, 20 to 50, 30 to 90, 20 to 100, 20 to60, 40 to 80, 40 to 100, 20 to 120, 20 to 40, 40 to 60, 60 to 80, 80 to100, 100 to 120 or 12 to 150 nucleotides long. In preferred embodiments,the oligonucleotide probe is 15 to 60 nucleotides in length.

When using a nucleic acid probe, all or only a portion of the probe canbe complementary to the target sequence. The portion of the probecomplementary to the target sequence is preferably at least 12nucleotides in length, and more preferably at least 15, at least 18 orat least 20 nucleotides in length. For nucleic acid probes of greaterlength than 40 or 50 nucleotides, the portion of the probe complementaryto the target sequence can be at least 25, at least 30 or at least 35nucleotides in length.

The antibody can be, for example, a polyclonal, monoclonal, or chimericantibody or an antigen binding fragment thereof (i.e., “antigen-bindingportion”) or single chain thereof, fusion proteins comprising anantibody, and any other modified configuration of the immunoglobulinmolecule that comprises an antigen recognition site, including, forexample without limitation, single chain (scFv) and domain antibodies(e.g., human, camelid, or shark domain antibodies), maxibodies,minibodies, intrabodies, diabodies, triabodies, tetrabodies, vNAR andbis-scFv (see e.g., Hollinger and Hudson, 2005, Nature Biotech23:1126-1136). An antibody includes an antibody of any class, such asIgG, IgA, or IgM (or sub-class thereof), and the antibody need not be ofany particular class. Depending on the antibody amino acid sequence ofthe constant domain of its heavy chains, immunoglobulins can be assignedto different classes. There are five major classes of immunoglobulins:IgA, IgD, IgE, IgG, and IgM, and several of these may be further dividedinto subclasses (isotypes), e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁ and IgA₂.“Antibody” also encompasses any of each of the foregoingantibody/immunoglobulin types.

Three-dimensional networks of the disclosure can comprise a singlespecies of probe or more than one species of probe (e.g., 2, 3, 4, or 5or more species). Three-dimensional networks can comprise more than onespecies of probe for the same target (e.g., antibodies binding differentepitopes of the same target) and/or comprise probes that bind multipletargets.

The networks can comprise a labeled (e.g., fluorescently labeled)control probe molecule that can be used, for example, to measure theamount probe present in the network.

The probes can be distributed throughout the network (e.g., on a surfaceand the interior of a network). Preferably, at least one probe is spacedaway from the surface of the network and adjoins at least one channel. Aprobe so located is then directly accessible for analyte molecules oranalyte components through the channel. In some embodiments, a majorityof the probes are located in the interior of the network.

The one or more probes can be immobilized on the network covalently ornon-covalently. For example, a probe can be crosslinked to thecrosslinked polymer or a probe can be non-covalently bound to thenetwork (such as by binding to a molecule covalently bound to thenetwork). In a preferred embodiment, one or more probes are crosslinkedto the crosslinked polymer. In some embodiments, a majority of theprobes are covalently bound in the interior of the network (e.g., suchthat at least a portion of the probes adjoin a channel).

Without being bound by theory, the inventors believe that the processesdescribed in Section 5.3 for manufacturing three-dimensional networks inthe presence of salt crystals (particularly phosphate salt crystals) mayresult in a greater concentration of probe molecule at or near theinterface between the polymer and the channel due to electrostaticinteractions between the probe molecules (particularly nucleic acidprobe molecules) and the salt crystals. Accordingly, in some embodimentsof the invention, the disclosure provides networks according to thedisclosure in which the probe density is greater at the interfacebetween the polymer and the channels than within regions of the polymernot abutting a channel. In various embodiments, the probe density it atleast 10%, at least 20%, at least 30%, at least 40%, or at least 50%more dense at the interface between the polymer and the channels thanwithin regions of the polymer not abutting a channel.

The density of probe molecule in a network can be verified using thefollowing procedure:

The network is brought into contact with an aqueous liquid at roomtemperature, for example, in a bowl. The liquid contains a plurality ofnanoparticles attached to a moiety that interacts with the probemolecules in the network, for example streptavidin if the probemolecules are biotinylated. The size of the nanoparticles is smallerthan the mesh size of the network and smaller than the minimumcross-section of the channel to allow the nanoparticles to becomedistributed throughout the polymer. Suitable nanoparticles are quantumdots 2-5 nanometers in diameter.

An incubation period is selected so that the network in the liquid iscompletely hydrated, i.e., that the network on average takes the sameamount of water as it releases. The incubation period can be, forexample, one hour. The penetration of the nanoparticles in the networkcan be accelerated by setting in motion the network and/or the liquidduring the incubation, for example, by vibrating the network and/orliquid, preferably by means of ultrasonic waves.

After completion of the incubation, the liquid is separated from thenetwork, for example, by draining the liquid from the bowl or taking thenetwork out of the bowl.

Then, the hydrated network is frozen, for example, by means of liquidnitrogen. Thereafter, the frozen network can be cut with the aid of acryomicrotome along mutually parallel cutting planes into thin slices.The cutting planes are arranged transversely to the longitudinalextension of the channel and penetrate the channel. The cutting ispreferably carried out using a liquid nitrogen-cooled diamond blade. Thethickness of the slices can be, for example, about 100 nm or 200 nm.

With the aid of a microscope, the nanoparticles disposed in the disksobtained by cutting the frozen network are located. The nanoparticlescan be fluorescent and optically highlighted so that they can be betterdistinguished from the network, if necessary. The locating of thenanoparticles can be done using a suitable software with imageprocessing methods. To examine the disks, preferably a confocalmicroscope laser scanning microscope with fluorescence optics or anelectron microscope is used.

The geometry and/or position information of the nanoparticles obtainedin this manner may be, with the aid of a computer, used to make athree-dimensional geometric model of distribution of the nanoparticlesin the network. The model can then be used to determine whether thedistribution of nanoparticles reflects a greater density of probemolecules near sites of channels.

5.2. Arrays

The three-dimensional networks of the disclosure can be positioned(e.g., deposited) on a substrate, and are preferably immobilized on asubstrate (e.g., by covalent crosslinks between the network and thesubstrate). A plurality of networks can be immobilized on a substrate toform an array useful, for example, as a biochip.

Suitable substrates include organic polymers, e.g., cycloolefincopolymers (COCs), polystyrene, polyethylene, polypropylene andpolymethylmethacrylate (PMMA, Plexiglas®). Ticona markets an example ofa suitable COC under the trade name Topas®. Inorganic materials (e.g.,metal, glass) can also be used as a substrate. Such substrates can becoated with organic molecules to allow for crosslinks between thenetwork and a surface of the substrate. For example, inorganic surfacescan be coated with self-assembled monolayers (SAMs). SAMs can themselvesbe completely unreactive and thus comprise or consist of, for example,pure alkyl silanes. Other substrates can also be suitable forcrosslinking to the three-dimensional network provided they are able toenter into stable bonds with organic molecules during free-radicalprocesses (e.g., organoboron compounds).

The substrate can be rigid or flexible. In some embodiments, thesubstrate is in the shape of a plate (e.g., a rectangular plate, asquare plate, a circular disk, etc.). For example, the substrate cancomprise a microwell plate, and the three-dimensional networks can bepositioned in the wells of the plate.

The individual networks can be positioned at distinct spots on a surfaceof the substrate, e.g., in a matrix comprising a plurality of columnsand rows. In the embodiment shown in FIG. 11, the networks are locatedat 36 spots arranged in six columns and six rows. Arrays havingdifferent numbers of rows and columns, the number of each of which canbe independently selected, are contemplated (e.g., 2 to 64 columns and 2to 64 rows). The columns can be separated by a distance X and the rowscan be separated by a distance Y (for example, as shown in FIG. 12) soas to form a grid of spots on which the individual networks can belocated. X and Y can be selected so that the networks, located at thespots of the grid, do not contact each other in the dehydrated state anddo not contact each other in the hydrated state. The dimensions X and Ycan be the same or different. In some embodiments, X and Y are the same.In some embodiments, X and Y are different. In some embodiments, X and Yare independently selected from distances of at least about 500 μm(e.g., 500 μm to 5 mm, 500 μm to 4 mm, 500 μm to 3 mm, 500 μm to 2 mm,or 500 μm to 1 mm). In some embodiments, X and Y are both about 500 μm.In other embodiments, X and Y are both 500 μm.

In some embodiments, substrate is band-shaped (for example, as shown inFIG. 13). The networks can be arranged as a single row extending in thelongitudinal direction of a band-shaped organic surface, or can bearranged as multiple rows extending in the longitudinal direction of theband-shaped surface. The rows and columns in such band-shaped arrays canhave grid dimensions X and Y as described above.

The individual networks can each cover an area of the surface of thearray that is circular or substantially circular. Typically, thediameter of the area on the surface of the array covered by theindividual networks (i.e., the spot diameter) is 80 μm to 1000 μm. Invarious embodiments, the spot diameter is 80 μm, 100 μm, 120 μm, 140 μm,160 μm, 180 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm,900 μm, or 1000 μm, or selected from a range bounded by any two of theforegoing embodiments, e.g., 80 μm to 200 μm, 100 μm to 120 μm, 120 μmto 140 μm, 120 μm to 180 μm, 140 μm to 160 μm, 160 μm to 180 μm, 180 μmto 200 μm, 120 μm to 200 μm, 100 μm to 400 μm, 160 μm to 600 μm, or 120μm to 700 μm, and so on and so forth. In a preferred embodiment, thediameter ranges from 100 μm to 200 μm or a subrange thereof.

The arrays of the disclosure typically have at least 8 individualthree-dimensional networks. In certain aspects, the arrays have at least16, at least 24, at least 48, at least 96, at least 128, at least 256,at least 512, or at least 1024 individual three-dimensional networks. Insome embodiments, the arrays of the disclosure have 24, 48, 96, 128,256, 512, 1024, 2048, 4096 or 8192 individual networks, or have a numberof three-dimensional networks selected from a range bounded any two ofthe foregoing embodiments, e.g., from 8 to 128, 8 to 512, 24 to 8192, 24to 4096, 48 to 2048, 96 to 512, 128 to 1024, 24 to 1024, 48 to 512, 96to 1024, or 128 to 512 three-dimensional networks, and so on and soforth. In a preferred embodiment, number of three-dimensional networkson an array ranges from 8 to 1024. In a particularly preferredembodiment, the number of three-dimensional networks on an array rangesfrom 25 to 400.

The individual networks which comprise the arrays of the disclosure canhave identical or different probes (e.g., each network can have a uniqueset of probes, multiple networks can have the same set of probes andother networks can have a different set or sets of probes, or all ofnetworks can have the same set of probes). For example, networksarranged in the same row of a matrix can comprise the same probes andthe networks arranged in different rows of the matrix can have differentprobes.

Typically, the individual networks on an array vary by no more than 20%,no more than 15%, no more than 10% or no more than 5% from one anotherby spot diameter and/or network volume.

In some embodiments, the arrays comprise one or more individual networks(e.g., spots on an array) with one or more control oligonucleotides orprobe molecules. The control oligonucleotides can be labelled, e.g.,fluorescently labelled, for use as a spatial control (for spatiallyorienting the array) and/or a quantifying the amount of probe moleculesbound to the networks, for example, when washing and reusing an array ofthe disclosure (i.e., as a “reusability control”). The spatial andreusability control probes (which can be the same or different probes)are referred to in Section 7.2 as a “landing light”, where the sameprobe is used for both purposes.

The same spot on the array or a different spot on the array can furtherinclude an unlabelled probe that is complementary to a known target.When used in a hybridization assay, determining the signal strength ofhybridization of the unlabelled probe to the labelled target candetermine the efficiency of the hybridization reaction. When anindividual network (i.e., a spot on an array) is used both as areusability and/or spatial control and a hybridization control, adifferent fluorescent moiety can be used to label the target moleculethan the fluorescent moiety of the reusability control or spatialcontrol probes.

In some embodiments, the arrays of the disclosure can be reused at least5 times, at least 10 times, at least 20 times, at least 30 times, atleast 40 times, or at least 50 times (e.g., 5 to 20 times, 5 to 30times, 10 to 50 times, 10 to 20 times, 10 to 30 times, 20 to 40 times,or 40 to 50 times, preferably comprising reusing the array 10 to 50times). The array can be washed with a salt solution under denaturatingconditions (e.g., low salt concentration and high temperature). Forexample, the array can be washed with a 1-10 mM phosphate buffer at80-90° C. between uses. The temperature of the wash can be selectedbased upon the length (Tm) of the target:probe hybrid.

The integrity of an array can be determined by a “reusability control”probe. The reusability control probe can be fluorescently labeled or canbe detected by hybridization to a fluorescently labeled complementarynucleic acid. The fluorescent label of a fluorescently labeledreusability control probe may be bleached by repeated excitation, beforethe integrity of the nucleic acid is compromised; in such cases anyfurther reuses can include detection of hybridization to a fluorescentlylabeled complementary nucleic acid as a control. Typically, an array ofthe invention is stable for at least 6 months.

In various embodiments, a fluorescently labeled reusability controlprobe retains at least 99%, 95% 90%, 80%, 70%, 60%, or 50% of itsinitial fluorescence signal strength after 5, 10, 20, 30, 40, or 50uses. Preferably, the reusability control probe retains least 75% of itsfluorescence signal strength after 5 or 10 uses. An array can continueto be reused until the reusability control probe retains at least 50% ofits fluorescence signal strength, for example after 20, 30, 40 or 50reuses. The fluorescent signal strength of the control probe can betested between every reuse, every other reuse, every third reuse, everyfourth reuse, every fifth reuse, every sixth reuse, every seventh reuse,every eighth reuse, every ninth reuse, every tenth reuse, or acombination of the above. For example, the signal strength can be testedperiodically between 5 or 10 reuses initially and the frequency oftesting increased with the number of reuses such that it is tested aftereach reuse after a certain number (e.g., 5, 10, 20, 30, 40 or 50) uses.In some embodiments, the frequency of testing averages once per 1, 1.5,2, 2.5, 3, 4, 5 or 10 uses, or averages within a range bounded betweenany two of the foregoing values, e.g., once per 1-2 uses, once per 1-1.5uses, once per 1-3 uses, or once per 1.5-3 uses.

It is noted that the nomenclature of “spatial control”, “reusabilitycontrol” and “hybridization control” is included for convenience andreference purposes and is not intended to connote a requirement that theprobes referred to “spatial control”, “reusability control” and“hybridization control” be used as such.

5.3. Processes for Making Three-Dimensional Polymer Networks

In one aspect, the processes of the disclosure for makingthree-dimensional polymer networks comprise (a) exposing a mixturecomprising an aqueous salt solution, a polymer, a cross-linker and,optionally, one or more probes to salt crystal forming conditions, (b)exposing the mixture to crosslinking conditions to crosslink the polymerfor form a crosslinked polymer network, and (c) contacting thecrosslinked polymer network with a solvent to dissolve the salt crystalsand form one or more channels.

The processes can further comprise a step of forming the mixture bycombining an aqueous salt solution, a polymer, a cross-linker and,optionally, one or more probes, and/or further comprise a step ofapplying the mixture to a substrate (e.g., a substrate described inSection 5.2) prior to exposing the mixture to salt forming conditions.If the polymer being used has a pre-attached cross-linker (e.g., whenusing a copolymer polymerized from a monomer comprising a cross-linker),the step of forming the mixture can comprise combining an aqueous saltsolution with the polymer and, optionally, one or more probes.

The channels formed by dissolution of the salt crystals can have one ormore of the features described in Section 5.1.3.

The mixture can be applied to a substrate prior to exposing the mixtureto salt forming conditions for example, by spraying the mixture onto asurface of the substrate (e.g., at 1024 sites on the surface). Themixture can be applied to the surface using a DNA chip spotter or inkjetprinter, for example. In a preferred embodiment, the mixture is sprayedusing an inkjet printer. This permits a simple and rapid application ofthe mixture to a large number of spots on the substrate. The spots canbe arranged, for example, in the form of a matrix in several rows and/orcolumns. Preferably, the salt content in the mixture during printing isbelow the solubility limit so that the mixture does not crystallize inthe printing head of the printer. The volume of mixture applied atindividual spots can be, for example, 100 pl, 200 pl, 300 pl, 400 pl,500 pl, 750 pl, 1 nl, 2 nl, 3 nl, 4 nl, or 5 nl, or can be selected froma range bounded by any two of the foregoing values (e.g., 100 pl to 5nl, 100 pl to 1 nl, 300 pl to 1 nl, 200 pl to 750 nl, 100 pl to 500 pl,200 pl to 2 nl, 500 pl to 2 nl 1 nl to 2 nl, and so on and so forth). Inpreferred embodiments, the spot volume is 200 pl to 4 nl.

The diameter of the individual spots will depend on the composition ofthe mixture, the volume of the mixture applied, and the surfacechemistry of the substrate. Spot diameters typically range between 80 μmto 1000 μm and can be obtained by varying the foregoing parameters. Invarious embodiments, the spot diameters are 80 μm, 100 μm, 120 μm, 140μm, 160 μm, 180 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800μm, 900 μm, or 1000 μm, or selected from a range bounded by any two ofthe foregoing embodiments, e.g., 80 μm to 200 μm, 100 μm to 120 μm, 120μm to 140 μm, 120 μm to 180 μm, 140 μm to 160 μm, 160 μm to 180 μm, 180μm to 200 μm, 120 μm to 200 μm, 100 μm to 400 μm, 160 μm to 600 μm, or120 μm to 700 μm, and so on and so forth. In a preferred embodiment, thediameter ranges from 100 μm to 200 μm or a subrange thereof.

Suitable polymers, cross-linkers, and probes that can be used in theprocesses of the disclosure are described in Sections 5.1.1, 5.1.2, and5.1.4, respectively. In some embodiments, the polymer used in theprocesses has at least one cross-linker group per polymer molecule. In apreferred embodiment, the polymer has at least two cross-linker groupsper molecule. In a particularly preferred embodiment, the polymer has atleast two photoreactive cross-linker groups per molecule. In theseembodiments, separate polymer and cross-linker molecules are not needed.

Suitable salts that can be included in the mixture are described inSection 5.3.1. Suitable salt forming conditions are described in Section5.3.2. Suitable crosslinking conditions are described in Section 5.3.3.Suitable solvents for dissolving the salt crystals are described inSection 5.3.4.

5.3.1. Salt

The salt can be selected for its compatibility with one or more probes.Ideally, the salt has one or more of the following characteristics, (i)the salt is not toxic to the probes (e.g., the salt does not denaturethe probes), (ii) the salt does not react chemically with the probes,(iii) the salt does not attack fluorophores, such as cyanine dyes, whichare suitable for the optical marking of probes, (iv) the salt does notreact with analytes, detection molecules, and/or binding partners bondedthereto, and/or (v) the salt forms needle-shaped crystals.

In a preferred embodiment, the salt solution comprises monovalentcations. The mixture can comprise disodium hydrogen phosphate and/orsodium dihydrogen phosphate which, in aqueous solution, releases Na⁺cations and phosphate ions PO₄ ³⁻. Sodium phosphate is readily solublein water and forms colorless crystals.

In a particularly preferred embodiment, the mixture comprisesdipotassium hydrogen phosphate (K₂HPO₄) and/or potassium dihydrogenphosphate (KH₂PO₄). These salts are excellently soluble in water and cantherefore form a correspondingly large number of needle-shaped saltcrystals in the mixture.

5.3.2. Salt Crystal Forming Conditions

Salt crystal forming conditions can comprise forming in the mixture atleast one salt crystal, preferably a needle-shaped salt crystal, bydehydrating the mixture or cooling the mixture until the relative saltcontent in the mixture increases to above the solubility limit, meaningthat the mixture is supersaturated with the salt. This promotes theformation of salt crystals from a crystallization germ located in thevolume of the mixture towards the surface of the mixture.

The mixture can be dehydrated by heating the mixture, exposing themixture to a vacuum, and/or reducing the humidity of the atmospheresurrounding the mixture.

The mixture can be heated by placing the mixture on a heated substrateor surface (e.g., between about 50° C. to about 70° C.), heating thesubstrate or surface on which the mixture has been placed (e.g., tobetween about 50° C. to about 70° C.), and/or contacting the mixturewith a hot gas (e.g., air, nitrogen, or carbon dioxide having atemperature that is higher than the temperature of the mixture) suchthat water is evaporated from the mixture. The contacting with the hotgas can, for example, take place by placing the mixture in a heatingoven. During the transportation to the heating oven, the mixture ispreferably kept moist, in particular at a relative humidity of above75%. As a result of this, an uncontrolled formation of salt crystalsduring the transportation of the mixture to the heating oven iscounteracted. This permits the formation of longer, needle-shaped saltcrystals in the heating oven. By heating the mixture it is also possibleto activate thermally activatable cross-linkers, if present, andcrosslink the polymer thereby.

In some embodiments, the temperature of the heated substrate and/or airused to dehydrate the mixture is 20° C. or more above the temperature ofthe mixture before heating the mixture, but less than 100° C.

The mixture can be cooled by placing the mixture on a cooled substrateor surface (e.g., between about 5° C. to about 15° C.), cooling thesubstrate or surface on which the mixture has been placed (e.g., tobetween about 5° C. to about 15° C.) and/or bringing it into contactwith a cold gas (e.g., air, nitrogen, or carbon dioxide having atemperature that is lower than the temperature of the mixture). Whencooled, the temperature-dependent solubility limit of the salt in themixture decreases until the mixture is ultimately supersaturated withthe salt. The formation one or more salt crystals, preferablyneedle-shaped, is promoted by this. In some embodiments, the mixture iscooled by incubating it in a cold chamber with low humidity (e.g.,temperatures between 0° C. and 10° C., relative humidity <40%)

The temperature in the mixture is preferably held above the dew point ofthe ambient air surrounding the mixture during the formation of the oneor more salt crystals. This prevents the mixture becoming diluted withwater condensed from the ambient air, which could lead to a decrease inthe relative salt content in the mixture.

5.3.3. Crosslinking Conditions

The crosslinking conditions can be selected based upon the type ofcross-linker used. For example, when using a cross-linker activated byultraviolet light (e.g., benzophenone, a thioxanthone or a benzoinether), the crosslinking conditions can comprise exposing the mixture toultraviolet (UV) light. In some embodiments, UV light having awavelength from about 250 nm to about 360 nm is used (e.g., 260±20 nm or355±20 nm). The use of lower energy/longer wavelength UV light (e.g.,360 nm UV light vs. 254 nm UV light) can require longer exposure times.When using a cross-linker activated by visible light (e.g., ethyl eosin,eosin Y, rose bengal, camphorquinone or erythrosin), the crosslinkingconditions can comprise exposing the mixture to visible light. Whenusing a thermally activated cross-linker (e.g., 4,4′azobis(4-cyanopentanoic) acid, and 2,2-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride, or benzoyl peroxide), the crosslinkingconditions can comprise exposing the mixture to heat.

The length and intensity of the crosslinking conditions can be selectedto effect crosslinking of polymer molecules to other polymer molecules,crosslinking of polymer molecules to probe molecules (if present), andcrosslinking of polymer molecules to substrate molecules or organicmolecules present on the substrate (if present). The length andintensity of crosslinking conditions for a mixture containing probes canbe determined experimentally to balance robustness of immobilization andnativity of probe molecules, for example.

5.3.4. Salt Crystal Dissolution

After crosslinking the polymer, the one or more salt crystals can bedissolved in the solvent in such a way that at least one channel isformed in the network, said channel extending starting from the surfaceand/or near the surface of the network into the interior of the network.Advantageously, after the salt crystals have dissolved in a solvent, ahollow, elongated channel is produced in the place where the saltcrystal was, according to the principle of the “lost” form. The channelsallow analytes to penetrate through the channel into the interior of thenetwork and specifically bind a probe located in the interior of thenetwork. When using an array produced by the method of the disclosure asa biological sensor, a high measurement accuracy and high measurementdynamic are permitted.

The solvent for dissolving the one or more salt crystals can be chosenin such a way that it is compatible to the polymer and probes, ifpresent (e.g., the solvent can be chosen such that it does not dissolvethe polymer and probes). Preferably, the solvent used is a water basedbuffer, such as diluted phosphate buffer. Methanol, ethanol, propanol ora mixture of these liquids can be added to the buffer to facilitate theremoval of unbound polymer from the network.

After the removal of the salt crystals the network can collapse due todrying and can be rehydrated. Drying the network has advantages forshipping and stabilization of probe biomolecules.

5.3.5. Methods of Using the Three-Dimensional Networks

The networks and arrays of the disclosure can be used to determine thepresence or absence of an analyte in a sample, preferably a liquidsample. The disclosure therefore provides methods for determiningwhether an analyte is present in a sample or plurality of samples,comprising contacting a network or array of the disclosure comprisingprobe molecules that are capable of binding to the analyte with thesample or plurality of samples and detecting binding of the analyte tothe probe molecules, thereby determining whether the analyte is presentin the sample or plurality of samples. When arrays comprising differentspecies of probes capable of binding different species of analyte areused in the methods, the presence of the different species of analytescan be determined by detecting the binding of the different species ofanalytes to the probes. In some embodiments, the methods furthercomprise a step of quantifying the amount of analyte or analytes boundto the array.

The analyte can be, for example, a nucleic acid, such as a polymerasechain reaction (PCR) amplicon. In some embodiments, the PCR amplicon isamplified from a biological or environmental sample (e.g., blood, serum,plasma, tissue, cells, saliva, sputum, urine, cerebrospinal fluid,pleural fluid, milk, tears, stool, sweat, semen, whole cells, cellconstituent, cell smear, or an extract or derivative thereof). In someembodiments, the nucleic acid is labeled (e.g., fluorescently labeled).

An analyte placed on the surface of the network can penetrate into theinterior of the network through the channel in order to specificallybind to a probe (e.g., a biomolecule) covalently bonded there to thepolymer. When using the arrays of the disclosure with the networksimmobilized thereon as biological sensor, a high measurement accuracyand also a high measurement dynamic is permitted.

The networks and arrays of the disclosure can be regenerated after useas a biosensor and can be used several times (e.g., at 5 times, at least10 times, at least 20 times, at least 30 times, at least 40 times, or atleast 50 times). If the probe molecules are DNA, this can be achieved,for example, by heating the network(s) in an 1× phosphate bufferedsaline to a temperature between 80° C. and 90° C. for about 10 minutes.Then, the phosphate buffered saline can be exchanged for a new phosphatebuffered saline to wash the denatured DNA out of the network(s). If theprobe molecules of the network(s) or array are antigens the network(s)or array can be regenerated by bringing the network(s) into contact with0.1N NaOH for about 10 minutes. Then, the 0.1N NaOH can be exchanged fora phosphate buffered saline to wash the antigens out of the network.Thus, some embodiments of the methods of using the networks and arraysof the disclosure comprise using a network or array that has been washedprior to contact with a sample or a plurality of samples.

5.4. Applications of Arrays of the Disclosure

Because the arrays of the invention achieve economical determination ofthe qualitative and quantitative presence of nucleic acids in a sample,it has immediate application to problems relating to health and diseasein human and non-human animals.

In these applications a preparation containing a target molecule isderived or extracted from biological or environmental sources accordingto protocols known in the art. The target molecules can be derived orextracted from cells and tissues of all taxonomic classes, includingviruses, bacteria and eukaryotes, prokaryotes, protista, plants, fungi,and animals of all phyla and classes. The animals can be vertebrates,mammals, primates, and especially humans. Blood, serum, plasma, tissue,cells, saliva, sputum, urine, cerebrospinal fluid, pleural fluid, milk,tears, stool, sweat, semen, whole cells, cell constituent, and cellsmears are suitable sources of target molecules.

The target molecules are preferably nucleic acids amplified (e.g., byPCR) from any of the foregoing sources).

The arrays of the invention can include probes that are useful to detectpathogens of humans or non-human animals. Such probes includeoligonucleotides complementary at least in part to bacterial, viral orfungal targets, or any combinations of bacterial, viral and fungaltargets.

The arrays of the invention can include probes useful to detect geneexpression in human or non-human animal cells, e.g., gene expressionassociated with a disease or disorder such as cancer, cardiovasculardisease, or metabolic disease for the purpose of diagnosing a subject,monitoring treatment of a subject or prognosis of a subject's outcome.Gene expression information can then track disease progression orregression, and such information can assist in monitoring the success orchanging the course of an initial therapy.

6. EXEMPLARY PROTOCOLS

The following exemplary protocols, which refer to the reference numbersprovided in the figures, are within the scope of the disclosure and canbe used in conjunction with the polymers, cross-linkers and probes ofSections 5.1.1, 5.1.2 and 5.1.4, respectively. Further useful polymers(including co-polymers) and cross-linker groups for use in the followingmethods are described in Rendl et al., 2011, Langmuir 27:6116-6123 andin US 2008/0293592, the contents of which are incorporated by referenceherein. In one embodiment, a polymer mixture according to Section 7.1 isused.

6.1. Exemplary Protocol 1

A plate with an organic surface (2) is placed on a heated holder (6).Temperatures between 50° C. and 70° C. are suitable. A mixture (5)containing a polymer (3), probe biomolecules (1) and an aqueous saltsolution is spotted on the organic surface (2) using a standard DNA chipspotter (e.g., Scienion, Germany). Volumes of 0.5 to 4 nl are printed oneach spot (7) (see, FIG. 2). The liquid of these spots dries almostimmediately leading to a very fast nucleation of salt crystals (8).After nucleation, needle-shaped salt crystals can extend from at leastone crystallization germ (9) located in the volume of the mixture (5) tothe surface (10) of the mixture (5) (see, FIG. 3). After nucleation ofthe crystals (8) the spots (7) are irradiated in a UV cross-linkerimmediately with optical UV radiation (11) (see, FIG. 4) such that theprobe biomolecules (1) are covalently bonded to the polymer (3), and thepolymer (3) is covalently bonded to the organic surface (2) andcrosslinked (see, FIG. 5). Care is taken that the dried, crosslinkedmixture (5) is not attracting moisture to become liquid again.

The dried, crosslinked mixture (5) is then brought into contact with asolvent (12) for the crystals (8) such that at the places at which thecrystals (8) were, channels (13) are formed in the network (15)comprising the polymer (3) and the probe biomolecules (1) (see, FIG. 6).Thereafter, the solvent (12) is removed. The channels (13) can extendfrom the surface (16) of the network (15) into the interior of thenetwork (15). The solvent (12) in which the salt crystals (8) aredissolved is chosen in such a way that it is compatible to the probebiomolecule (1) and also the polymer (3). Preferably, the solvent (12)used is water based.

6.2. Exemplary Protocol 2

A mixture (5) containing a polymer (3), probe biomolecules (1) and anaqueous salt solution is spotted on an organic surface (2) arranged on aplate using a standard DNA chip spotter (e.g., Scienion, Germany).Volumes of 0.5 to 4 nl are printed on each spot 7 (see, FIG. 2). Theplate with the spots (7) on the organic surface (2) is placed on achilled holder (14) (see, FIG. 7). Temperatures between 5° C. and 15° C.are suitable. The liquid of these spots is cooled down to reach an oversaturation of the buffer that almost immediately leads to a nucleationof crystals. After nucleation needle-shaped salt crystals (8) can extendfrom at least one crystallization germ (9) located in the volume of themixture (5) to the surface (10) of the mixture (5). After printing thesetargets are put in an oven (e.g., at 70° C.) for complete drying. Afternucleation of the crystals the spots are irradiated in a UV cross-linkerimmediately with optical UV radiation (11) (see, FIG. 8) such that theprobe biomolecules (1) are covalently bonded to the polymer (3), and thepolymer (3) is covalently bonded to the organic surface (2) andcrosslinked. Care is taken that the dried, crosslinked mixture is notattracting moisture to become liquid again.

The dried, crosslinked mixture (5) is then brought into contact with asolvent (12) to dissolve the crystals (8) such that at the places atwhich the crystals (8) were, channels (13) are formed in the network(15) comprising the polymer (3) and the probe biomolecules (1).Thereafter, the solvent (12) is removed. The channels (13) can extendfrom the surface (16) of the network (15) into the interior of thenetwork (15). The solvent (12) in which the salt crystals (8) aredissolved is chosen in such a way that it is compatible with the probebiomolecule (1) and the polymer (3). Preferably, the solvent (12) usedis water based.

As can be seen in FIG. 9 a plurality of channels (13) can be formed inthe network (15). The channels (13) can extend from the surface (16) ofthe network (15) to at least one point located within the network (15).The channels (13) can be arranged in such a way that—starting from thesurface (16) in the direction of the interior—the lateral distancebetween the channels (13) decreases.

6.3. Exemplary Protocol 3

A mixture (5) containing a polymer (3), probe biomolecules (1) and anaqueous salt solution is printed on an organic surface (2) of a plate atnormal conditions with a humidity ranging from 40-80%, preferably50-70%. The mixture can be near saturation, 400 mM sodium phosphate, pH8, for example. Volumes of 0.5 to 4 nl are printed on each spot (7). Themoisture content in the print compartment makes sure the spots (7) stayliquid without crystal formation (i.e., no nucleation takes place). Theplate is then put in a container, a cardboard box for example. Lids areput on the plate having the organic surface (2) for transport. The platewith the spots (7) on the organic surface (2) is then put in a dryingoven or on a hot plate to rapidly cause nucleation such thatneedle-shaped salt crystals (8) extend from at least one crystallizationgerm (9) located in the volume of the mixture toward the surface 10 ofthe mixture (5).

The temperature of the oven/hot plate should be 20° C. or more above theprinting temperature. Temperatures above 100° C. are not necessary.

After drying, the mixture is irradiated to crosslink the polymer (3),probe biomolecules (1), and organic surface (2).

The dried, crosslinked mixture (5) is then brought into contact with asolvent (12) such that at the places at which the crystals (8) were,channels (13) are formed in the network (15) comprising the polymer (3)and the probe biomolecules (1). Thereafter, the solvent (12) is removed.The channels (13) can are extend from the surface (16) of the network(15) into the interior of the network (15). The solvent (12) in whichthe salt crystals (8) are dissolved is chosen in such a way that it iscompatible with the probe biomolecules (1) and the polymer (3).Preferably, the solvent (12) used is water based.

6.4. Exemplary Protocol 4

Alternatively, a plate with spots (7) on the organic surface (2)prepared as in exemplary protocol 3 can be cooled to achieve nucleationby putting in a cold chamber with low humidity (e.g., temperatures <10°C., relative humidity <40%). The drying can be performed by reducing thehumidity or by applying a vacuum after nucleation has started. Afternucleation, needle-shaped salt crystals (8) can extend from at least onecrystallization germ (9) located in the volume of the mixture (5) towardthe surface (10) of the mixture (5). The plate with the spots (7) on theorganic surface (2) is put in an oven at 60°−70° C. for 1 hour to fullydry the spots. The spots (7) are UV irradiated with 1.00 J @ 254 nm in aUV cross-linker, i.e. Stratalinker 2400. To do this, the plate with thespots (7) on the organic surface (2) can be put into the center of thechamber with the shorter side parallel to the door of the chamber. Then,the cover is removed and the cross-linker is started. When machine isfinished the array is removed and the cover is replaced.

Alternatively, other UV cross-linkers with the same wavelength (240-270nm, for example) or longer wavelengths, e.g., 360 nm, can be used.

The mixture (5) is then brought into contact with a solvent (12) todissolve the crystals (8) such that at the places at which the crystals(8) were, channels (13) are formed in the network (15) comprising thepolymer (3) and the probe biomolecules (1). Thereafter, the solvent (12)is removed. The channels (13) can extend from the surface (16) of thenetwork (15) into the interior of the network (15). The solvent (12) inwhich the salt crystals (8) are dissolved is chosen in such a way thatit is compatible with the probe biomolecules (1) the polymer (3).Preferably, the solvent (12) used is water based.

7. EXAMPLES 7.1. Example 1: Formation of a Three-Dimensional PolymerNetwork with Channels

A 10 mg/ml polymer stock solution is prepared by dissolving 10 mg of thecrosslinking polymer poly(dimethylacrylamide) co 5%methacryloyloxybenzophenone co 2.5% Sodium 4-vinylbenzenesulfonate in1.0 ml of DNAse free water. This is achieved by vigorous shaking andvortexing for approximately 5 minutes until all the visible polymer isdissolved. The stock solution is then wrapped in foil to protect it fromlight and placed in a refrigerator overnight to ensure the polymercompletely dissolves and to allow the foam to reduce. The polymer has atleast two photoreactive groups per molecule.

A mixture comprising the polymer, DNA oligonucleotide probes, and sodiumphosphate is made by mixing 10 μl of a 100 μM DNA oligonucleotide stocksolution, 5 μl of the 10 mg/ml polymer stock solution (to provide aconcentration of polymer in the mixture of 1 mg/ml), and 35 μl of a 500mM sodium phosphate buffer, pH 8.

The mixture is used to prepare a three-dimensional network of theinvention using the method of any one of Exemplary Protocols 1 through4.

7.2. Example 2: Use of a Three-Dimensional Polymer Network to DetectBacteria 7.2.1. Preparation of an Array of the Invention

Oligonucleotides for immobilization were dissolved at a concentration of20 μM in a 400 mM sodium phosphate buffer, pH 7 containing 1 mg/ml ofthe photoreactive polymer described in Example 1. Each oligonucleotidehad a length of 30-35 nucleotides complementary to the target DNA and atail of 15 thymidines (for a total oligonucleotide length of 45-50nucleotides).

The mixture was used to print the following spots on an organic surfaceof a plate to provide an array:

-   -   LL: So called landing lights. Cy5-labelled DNA oligonucleotide        (0.2 μM), polymer and unlabeled oligonucleotide 19.8 μM to make        up to 20 μM total oligonucleotide concentration.    -   GN: Oligonucleotide specific to gram negative bacteria.    -   GP: Oligonucleotide specific to gram positive bacteria.    -   S.Aure_1: Oligonucleotide specific to Staphylococcus aureus        bacteria.    -   S.Aure_2: Oligonucleotide specific to Staphylococcus aureus        bacteria.    -   E. coli_1: Oligonucleotide specific to Escherichia coli        bacteria.    -   E. coli_2: Oligonucleotide specific to Escherichia coli        bacteria.

In order to avoid the formation of salt crystals on the source plate(i.e., the plate from which the mixture was taken) this plate was keptat ambient temperature (22° C.).

A Greiner 96 well plate with a flat crystal clear bottom with an organicsurface was cooled to 10° C. to avoid drying out of the printed spots.Using a Scienion® SciFlex 5 printer with a PDC 90 nozzle 8 drops perspot were printed on the organic surface, resulting in a spot volume ofapprox. 1.4 nl. The humidity of the printer was kept at 60-65% relativehumidity.

After the print the size of the spots was checked by an automated headcamera on the print head to assure that no drying in or crystalformation has taken place in the spots. All spots were still wet andhave the same size. No crystal formation was visible.

The 96 well plate was then sealed with a lid to avoid drying in andimmediately put on a hot plate (70° C.) in a drying oven to performcrystal initiation and drying of the spots.

After 1 hr incubation at 70° C. to ensure proper drying of the spots theplate was irradiated with 1J @ 254 nm in a Stratalinker® 2000.

7.2.2. Preparation of a Control Array

A procedure similar to that described in Section 7.2.1 was used but thetarget plate was kept at ambient temperature substantially as describedby Rendl et al., 2011, Langmuir 27:6116-6123. After the print, somespots showed a reduced size, i.e., some of the spots in random places inthe array were dried in and exhibited phase separation immediately afterthe printing. The plate was then taken out of the printer and taken tothe sample drying process as described above with no lid on, upon whichfurther drying in occurred.

7.2.3. Hybridization Assay

Before use the plates were washed in a plate washer for 3 times with 300μl of wash buffer (100 mM sodium phosphate pH 7) and then the buffer wasexchanged to 1 mM sodium phosphate pH 7. The plates were heated for 10minutes at 90° C. on a heater shaker to extract unbound DNA and polymer.The buffer then was exchanged to 100 mM sodium phosphate buffer using anautomated 96 well plate washer.

A mix of 20 ml Cy5-labeled PCR product and 30 ml sodium phosphate buffer(250 mM, pH7) was incubated on the arrays for 10 minutes at 80° C. and30 minutes at 55° C. on heater shaker. Afterward the plates were washedwith 100 mM sodium phosphate buffer pH7 for three times in 96 well platewasher. Plates with buffer were scanned in Sensovation® Flair reader andthe spot intensity of the different spots was measured.

7.2.4. Results

8 arrays produced by the method of Section 7.2.1 and 8 arrays producedby the method of Section 7.2.2 were analysed and the data processed in aspreadsheet program. Mean and standard error of the mean (SEM) werecalculated and compared. Results are shown in FIGS. 14-16.

8. EXAMPLE 3: REUSABILITY OF A THREE-DIMENSIONAL POLYMER NETWORK ARRAY

Several arrays were prepared according to the procedure described inSection 7.2.1, including a “landing light” spot containing fluorescentlylabeled oligonucleotides. The arrays were reused in hybridization assaysand washed according to the following procedure in betweenhybridizations:

(a) the arrays were washed three times with 100 mM phosphate buffer pH7;

(b) the buffer was then exchanged to 1 mM phosphate buffer pH 7; and

(c) the arrays were then heated to 80° C. and washed while hot with 100mM phosphate buffer pH 7.

The strength of the fluorescent signal from the landing light spot wasdetermined in between uses. After 10 uses, the fluorescent signal lostless than 25% of its intensity. The arrays were reused until a referencespot hybridized with a reference DNA (internal control) showed a loss ofsignal of 50%.

9. SPECIFIC EMBODIMENTS

The present disclosure is exemplified by the specific embodiments below.

1. A three-dimensional network having a surface and an interiorcomprising (a) a crosslinked polymer, (b) one or more channels, and (c)optionally, probe molecules immobilized in the network, saidthree-dimensional network optionally covalently attached to the surfaceof a substrate.

2. The three-dimensional network of embodiment 1, wherein at least oneof the one or more channels extends into the interior from a point thatis less than 10 microns from the surface of the network.

3. The three-dimensional network of embodiment 2, wherein at least oneof the one or more channels extends into the interior from a point thatis less than 9 microns from the surface of the network.

4. The three-dimensional network of embodiment 3, wherein at least oneof the one or more channels extends into the interior from a point thatis less than 8 microns from the surface of the network.

5. The three-dimensional network of embodiment 4, wherein at least oneof the one or more channels extends into the interior from a point thatis less than 7 microns from the surface of the network.

6. The three-dimensional network of embodiment 5, wherein at least oneof the one or more channels extends into the interior from a point thatis less than 6 microns from the surface of the network.

7. The three-dimensional network of embodiment 6, wherein at least oneof the one or more channels extends into the interior from a point thatis less than 5 microns from the surface of the network.

8. The three-dimensional network of embodiment 7, wherein at least oneof the one or more channels extends into the interior from a point thatis less than 4 microns from the surface of the network.

9. The three-dimensional network of embodiment 8, wherein at least oneof the one or more channels extends into the interior from a point thatis less than 3 microns from the surface of the network.

10. The three-dimensional network of embodiment 9, wherein at least oneof the one or more channels extends into the interior from a point thatis less than 2 microns from the surface of the network.

11. The three-dimensional network of embodiment 10, wherein at least oneof the one or more channels extends into the interior from a point thatis less than 1 micron from the surface of the network.

12. The three-dimensional network of embodiment 11, wherein at least oneof the one or more channels extends into the interior from a point onthe surface of the network.

13. The three-dimensional network of any one of embodiments 1 to 12,wherein at least one of the one or more channels has a length that is atleast 10% of the largest dimension of the network.

14. The three-dimensional network of embodiment 13, wherein at least oneof the one or more channels has a length that is 10% to 100% of thelargest dimension of the network.

15. The three-dimensional network of embodiment 13 or embodiment 14,wherein at least one of the one or more channels has a length that is atleast 15% of the largest dimension of the network.

16. The three-dimensional network of embodiment 13 or embodiment 14,wherein at least one of the one or more channels has a length that is atleast 20% of the largest dimension of the network.

17. The three-dimensional network of embodiment 13 or embodiment 14wherein at least one of the one or more channels has a length that is atleast 25% of the largest dimension of the network.

18. The three-dimensional network of embodiment 13 or embodiment 14,wherein at least one of the one or more channels has a length that is10% to 90% of the largest dimension of the network.

19. The three-dimensional network of embodiment 13 or embodiment 14,wherein at least one of the one or more channels has a length that is10% to 80% of the largest dimension of the network.

20. The three-dimensional network of embodiment 13 or embodiment 14,wherein at least one of the one or more channels has a length that is10% to 70% of the largest dimension of the network.

21. The three-dimensional network of embodiment 13 or embodiment 14,wherein at least one of the one or more channels has a length that is10% to 60% of the largest dimension of the network.

22. The three-dimensional network of embodiment 13 or embodiment 14,wherein at least one of the one or more channels has a length that is10% to 50% of the largest dimension of the network

23. The three-dimensional network of embodiment 13 or embodiment 14,wherein at least one of the one or more channels has a length that is10% to 40% of the largest dimension of the network

24. The three-dimensional network of embodiment 13 or embodiment 14,wherein at least one of the one or more channels has a length that is10% to 30% of the largest dimension of the network.

25. The three-dimensional network of embodiment 13 or embodiment 14,wherein at least one of the one or more channels has a length that is10% to 25% of the largest dimension of the network

26. The three-dimensional network of embodiment 13 or embodiment 14,wherein at least one of the one or more channels has a length that is10% to 20% of the largest dimension of the network

27. The three-dimensional network of embodiment 13 or embodiment 14,wherein at least one of the one or more channels has a length that is10% to 15% of the largest dimension of the network.

28. The three-dimensional network of embodiment 13 or embodiment 14,wherein at least one of the one or more channels has a length that is15% to 20% of the largest dimension of the network.

29. The three-dimensional network of embodiment 13 or embodiment 14,wherein at least one of the one or more channels has a length that is15% to 25% of the largest dimension of the network.

30. The three-dimensional network of embodiment 13 or embodiment 14,wherein at least one of the one or more channels has a length that is15% to 35% of the largest dimension of the network.

31. The three-dimensional network of embodiment 13 or embodiment 14,wherein at least one of the one or more channels has a length that is20% to 25% of the largest dimension of the network.

32. The three-dimensional network of embodiment 13 or embodiment 14,wherein at least one of the one or more channels has a length that is25% to 30% of the largest dimension of the network.

33. The three-dimensional network of any one of embodiments 1 to 32,wherein at least one of the one or more channels has a minimumcross-section of at least 5 times the network's mesh size.

34. The three-dimensional network of embodiment 33, wherein at least oneof the one or more channels has a minimum cross-section of at least 10times the network's mesh size.

35. The three-dimensional network of embodiment 34, wherein at least oneof the one or more channels has a minimum cross-section of at least 15times the network's mesh size.

36. The three-dimensional network of embodiment 35, wherein at least oneof the one or more channels has a minimum cross-section of at least 20times the network's mesh size.

37. The three-dimensional network of embodiment 33, wherein at least oneof the one or more channels has a minimum cross-section of 5 to 10 timesthe network's mesh size.

38. The three-dimensional network of embodiment 33, wherein at least oneof the one or more channels has a minimum cross-section of 5 to 15 timesthe network's mesh size.

39. The three-dimensional network of embodiment 33, wherein at least oneof the one or more channels has a minimum cross-section of 5 to 20 timesthe network's mesh size.

40. The three-dimensional network of embodiment 33, wherein at least oneof the one or more channels has a minimum cross-section of 5 to 25 timesthe network's mesh size.

41. The three-dimensional network of embodiment 33, wherein at least oneof the one or more channels has a minimum cross-section of 5 to 30 timesthe network's mesh size.

42. The three-dimensional network of embodiment 33, wherein at least oneof the one or more channels has a minimum cross-section of 10 to 15times the network's mesh size.

43. The three-dimensional network of embodiment 33, wherein at least oneof the one or more channels has a minimum cross-section of 10 to 20times the network's mesh size.

44. The three-dimensional network of embodiment 33, wherein at least oneof the one or more channels has a minimum cross-section of 10 to 25times the network's mesh size.

45. The three-dimensional network of embodiment 33, wherein at least oneof the one or more channels has a minimum cross-section of 10 to 30times the network's mesh size.

46. The three-dimensional network of embodiment 33, wherein at least oneof the one or more channels has a minimum cross-section of 15 to 20times the network's mesh size.

47. The three-dimensional network of embodiment 33, wherein at least oneof the one or more channels has a minimum cross-section of 15 to 25times the network's mesh size.

48. The three-dimensional network of embodiment 33, wherein at least oneof the one or more channels has a minimum cross-section of 15 to 30times the network's mesh size.

49. The three-dimensional network of embodiment 33, wherein at least oneof the one or more channels has a minimum cross-section of 20 to 25times the network's mesh size.

50. The three-dimensional network of embodiment 33, wherein at least oneof the one or more channels has a minimum cross-section of 20 to 30times the network's mesh size.

51. The three-dimensional network of embodiment 33, wherein at least oneof the one or more channels has a minimum cross-section of 25 to 30times the network's mesh size.

52. The three-dimensional network of any one of embodiments 1 to 51,comprising a plurality of channels.

53. The three-dimensional network of embodiment 52, wherein theplurality of channels is at least 5 channels.

54. The three-dimensional network of embodiment 53, wherein theplurality of channels is at least 10 channels.

55. The three-dimensional network of embodiment 54, wherein theplurality of channels is at least 15 channels.

56. The three-dimensional network of any one of embodiments 52 to 55,wherein the plurality of channels is up to 50 channels.

57. The three-dimensional network of embodiment 52, wherein theplurality of channels is 10 to 50 channels.

58. The three-dimensional network of any one of embodiments 52 to 57,wherein a plurality of channels converge at a point in the interior ofthe network such that the lateral distance between the channelsdecreases from the surface toward the point in the interior.

59. The three dimensional network of embodiment 58, wherein theplurality of channels are connected at their point of convergence.

60. The three dimensional network of embodiment 58, wherein a majorityof channels in the network converge at one or more points in theinterior of the network such that the lateral distance between thechannels decreases from the surface toward the point in the interior.

61. The three dimensional network of embodiment 60, wherein a pluralityof channels that converge at the same point in the interior of thenetwork are connected at their point of convergence.

62. The three dimensional network of embodiment 60, wherein the majorityof channels in the network are connected to other channels at one ormore convergence points in the network.

63. The three-dimensional network of any one of embodiments 52 to 62,wherein at least a plurality of channels, or wherein a majority of thechannels in the network, extend into the interior from a point that isless than 10 microns from the surface of the network.

64. The three-dimensional network of embodiment 63, wherein at least aplurality of channels, or wherein a majority of the channels in thenetwork, extend into the interior from a point that is less than 9microns from the surface of the network.

65. The three-dimensional network of embodiment 64, wherein at least aplurality of channels, or wherein a majority of the channels in thenetwork, extend into the interior from a point that is less than 8microns from the surface of the network.

66. The three-dimensional network of embodiment 65 wherein at least aplurality of channels, or wherein a majority of the channels in thenetwork, extend into the interior from a point that is less than 7microns from the surface of the network.

67. The three-dimensional network of embodiment 66, wherein at least aplurality of channels, or wherein a majority of the channels in thenetwork, extend into the interior from a point that is less than 6microns from the surface of the network.

68. The three-dimensional network of embodiment 67, wherein at least aplurality of channels, or wherein a majority of the channels in thenetwork, extend into the interior from a point that is less than 5microns from the surface of the network.

69. The three-dimensional network of embodiment 68, wherein at least aplurality of channels, or wherein a majority of the channels in thenetwork, extend into the interior from a point that is less than 4microns from the surface of the network.

70. The three-dimensional network of embodiment 69, wherein at least aplurality of channels, or wherein a majority of the channels in thenetwork, extend into the interior from a point that is less than 3microns from the surface of the network.

71. The three-dimensional network of embodiment 70, wherein at least aplurality of channels, or wherein a majority of the channels in thenetwork, extend into the interior from a point that is less than 2microns from the surface of the network.

72. The three-dimensional network of embodiment 71, wherein at least aplurality of channels, or wherein a majority of the channels in thenetwork, extend into the interior from a point that is less than 1micron from the surface of the network.

73. The three-dimensional network of embodiment 72, wherein at least aplurality of channels, or wherein a majority of the channels in thenetwork, extend into the interior from a point on the surface of thenetwork.

74. The three-dimensional network of any one of embodiments 52 to 73,wherein at least a plurality of channels, or wherein a majority of thechannels in the network, have a length that is at least 10% of thelargest dimension of the network.

75. The three-dimensional network of embodiment 74, wherein at least aplurality of channels, or wherein a majority of the channels in thenetwork, have a length that is 10% to 100% of the largest dimension ofthe network.

76. The three-dimensional network of embodiment 74 or embodiment 75,wherein at least a plurality of channels, or wherein a majority of thechannels in the network, have a length that is at least 15% of thelargest dimension of the network.

77. The three-dimensional network of embodiment 74 or embodiment 75,wherein at least a plurality of channels, or wherein a majority of thechannels in the network, have a length that is at least 20% of thelargest dimension of the network.

78. The three-dimensional network of embodiment 74 or embodiment 75,wherein at least a plurality of channels, or wherein a majority of thechannels in the network, have a length that is at least 25% of thelargest dimension of the network.

79. The three-dimensional network of embodiment 74 or embodiment 75,wherein at least a plurality of channels, or wherein a majority of thechannels in the network, have a length that is 10% to 90% of the largestdimension of the network.

80. The three-dimensional network of embodiment 74 or embodiment 75,wherein at least a plurality of channels, or wherein a majority of thechannels in the network, have a length that is 10% to 80% of the largestdimension of the network.

81. The three-dimensional network of embodiment 74 or embodiment 75,wherein at least a plurality of channels, or wherein a majority of thechannels in the network, have a length that is 10% to 70% of the largestdimension of the network.

82. The three-dimensional network of embodiment 74 or embodiment 75,wherein at least a plurality of channels, or wherein a majority of thechannels in the network, have a length that is 10% to 60% of the largestdimension of the network.

83. The three-dimensional network of embodiment 74 or embodiment 75,wherein at least a plurality of channels, or wherein a majority of thechannels in the network, have a length that is 10% to 50% of the largestdimension of the network

84. The three-dimensional network of embodiment 74 or embodiment 75,wherein at least a plurality of channels, or wherein a majority of thechannels in the network, have a length that is 10% to 40% of the largestdimension of the network

85. The three-dimensional network of embodiment 74 or embodiment 75,wherein at least a plurality of channels, or wherein a majority of thechannels in the network, have a length that is 10% to 30% of the largestdimension of the network.

86. The three-dimensional network of embodiment 74 or embodiment 75,wherein at least a plurality of channels, or wherein a majority of thechannels in the network, have a length that is 10% to 25% of the largestdimension of the network

87. The three-dimensional network of embodiment 74 or embodiment 75,wherein at least a plurality of channels, or wherein a majority of thechannels in the network, have a length that is 10% to 20% of the largestdimension of the network

88. The three-dimensional network of embodiment 74 or embodiment 75,wherein at least a plurality of channels, or wherein a majority of thechannels in the network, have a length that is 10% to 15% of the largestdimension of the network.

89. The three-dimensional network of embodiment 74 or embodiment 75,wherein at least a plurality of channels, or wherein a majority of thechannels in the network, have a length that is 15% to 20% of the largestdimension of the network.

90. The three-dimensional network of embodiment 74 or embodiment 75,wherein at least a plurality of channels, or wherein a majority of thechannels in the network, have a length that is 15% to 25% of the largestdimension of the network.

91. The three-dimensional network of embodiment 74 or embodiment 75,wherein at least a plurality of channels, or wherein a majority of thechannels in the network, have a length that is 15% to 30% of the largestdimension of the network.

92. The three-dimensional network of embodiment 74 or embodiment 75,wherein at least a plurality of channels, or wherein a majority of thechannels in the network, have a length that is 20% to 25% of the largestdimension of the network.

93. The three-dimensional network of embodiment 74 or embodiment 75,wherein at least a plurality of channels, or wherein a majority of thechannels in the network, have a length that is 25% to 30% of the largestdimension of the network.

94. The three-dimensional network of any one of embodiments 52 to 93,wherein at least a plurality of channels, or wherein a majority of thechannels in the network, have a minimum cross-section of at least 5times the network's mesh size.

95. The three-dimensional network of embodiment 94, wherein at least aplurality of channels, or wherein a majority of the channels in thenetwork, have a minimum cross-section of at least 10 times the network'smesh size.

96. The three-dimensional network of embodiment 95, wherein at least aplurality of channels, or wherein a majority of the channels in thenetwork, have a minimum cross-section of at least 15 times the network'smesh size.

97. The three-dimensional network of embodiment 96, wherein at least aplurality of channels, or wherein a majority of the channels in thenetwork, have a minimum cross-section of at least 20 times the network'smesh size.

98. The three-dimensional network of embodiment 94, wherein at least aplurality of channels, or wherein a majority of the channels in thenetwork, have a minimum cross-section of 5 to 10 times the network'smesh size.

99. The three-dimensional network of embodiment 94, wherein at least aplurality of channels, or wherein a majority of the channels in thenetwork, have a minimum cross-section of 5 to 15 times the network'smesh size.

100. The three-dimensional network of embodiment 94, wherein at least aplurality of channels, or wherein a majority of the channels in thenetwork, have a minimum cross-section of 5 to 20 times the network'smesh size.

101. The three-dimensional network of embodiment 94, wherein at least aplurality of channels, or wherein a majority of the channels in thenetwork, have a minimum cross-section of 5 to 25 times the network'smesh size.

102. The three-dimensional network of embodiment 94, wherein at least aplurality of channels, or wherein a majority of the channels in thenetwork, have a minimum cross-section of 5 to 30 times the network'smesh size.

103. The three-dimensional network of embodiment 94, wherein at least aplurality of channels, or wherein a majority of the channels in thenetwork, have a minimum cross-section of 10 to 15 times the network'smesh size.

104. The three-dimensional network of embodiment 94, wherein at least aplurality of channels, or wherein a majority of the channels in thenetwork, have a minimum cross-section of 10 to 20 times the network'smesh size.

105. The three-dimensional network of embodiment 94, wherein at least aplurality of channels, or wherein a majority of the channels in thenetwork, have a minimum cross-section of 10 to 25 times the network'smesh size.

106. The three-dimensional network of embodiment 94, wherein at least aplurality of channels, or wherein a majority of the channels in thenetwork, have a minimum cross-section of 10 to 30 times the network'smesh size.

107. The three-dimensional network of embodiment 94, wherein at least aplurality of channels, or wherein a majority of the channels in thenetwork, have a minimum cross-section of 15 to 20 times the network'smesh size.

108. The three-dimensional network of embodiment 94, wherein at least aplurality of channels, or wherein a majority of the channels in thenetwork, have a minimum cross-section of 15 to 25 times the network'smesh size.

109. The three-dimensional network of embodiment 94, wherein at least aplurality of channels, or wherein a majority of the channels in thenetwork, have a minimum cross-section of 15 to 30 times the network'smesh size.

110. The three-dimensional network of embodiment 94, wherein at least aplurality of channels, or wherein a majority of the channels in thenetwork, have a minimum cross-section of 20 to 25 times the network'smesh size.

111. The three-dimensional network of embodiment 94, wherein at least aplurality of channels, or wherein a majority of the channels in thenetwork, have a minimum cross-section of 20 to 30 times the network'smesh size.

112. The three-dimensional network of embodiment 94, wherein at least aplurality of channels, or wherein a majority of the channels in thenetwork, have a minimum cross-section of 25 to 30 times the network'smesh size.

113. The three-dimensional network of any one of embodiments 1 to 112,wherein the network has a mesh size of 5 nm to 75 nm in its hydratedstate.

114. The three-dimensional network of embodiment 113, wherein thenetwork has a mesh size of 10 nm to 50 nm in its hydrated state.

115. The three-dimensional network of embodiment 113, wherein thenetwork has a mesh size of 10 nm to 40 nm in its hydrated state.

116. The three-dimensional network of embodiment 113, wherein thenetwork has a mesh size of 10 nm to 30 nm in its hydrated state.

117. The three-dimensional network of embodiment 113, wherein thenetwork has a mesh size of 10 nm to 20 nm in its hydrated state.

118. The three-dimensional network of embodiment 113, wherein thenetwork has a mesh size of 20 nm to 50 nm in its hydrated state.

119. The three-dimensional network of embodiment 113, wherein thenetwork has a mesh size of 20 nm to 40 nm in its hydrated state.

120. The three-dimensional network of embodiment 113, wherein thenetwork has a mesh size of 20 nm to 30 nm in its hydrated state.

121. The three-dimensional network of embodiment 113, wherein thenetwork has a mesh size of 30 nm to 50 nm in its hydrated state.

122. The three-dimensional network of embodiment 113, wherein thenetwork has a mesh size of 30 nm to 40 nm in its hydrated state.

123. The three-dimensional network of embodiment 113, wherein thenetwork has a mesh size of 40 nm to 50 nm in its hydrated state.

124. The three-dimensional network of any one of embodiments 1 to 123,wherein the crosslinked polymer is a water-swellable polymer.

125. The three-dimensional network of embodiment 124, wherein thewater-swellable polymer can absorb up to 50 times its weight ofdeionized, distilled water.

126. The three-dimensional network of embodiment 124 or embodiment 125,wherein the water-swellable polymer can absorb 5 to 50 times its ownvolume of deionized, distilled water.

127. The three-dimensional network of any one of embodiments 124 to 126,wherein the water-swellable polymer can absorb up to 30 times its weightof saline.

128. The three-dimensional network of any one of embodiments 124 to 127,wherein the water-swellable polymer can absorb 4 to 30 times its ownvolume of saline.

129. The three-dimensional network of any one of embodiments 1 to 128,wherein the crosslinked polymer comprises a crosslinked homopolymer.

130. The three-dimensional network of any one of embodiments 1 to 128,wherein the crosslinked polymer comprises a crosslinked copolymer.

131. The three-dimensional network of any one of embodiments 1 to 128,wherein the crosslinked polymer comprises a crosslinked mixture of ahomopolymer and a copolymer.

132. The three-dimensional network of any one of embodiments 129 to 131,wherein the crosslinked polymer comprises a polymer polymerized from oneor more species of monomers.

133. The three-dimensional network of embodiment 132, wherein eachspecies of monomer comprises a polymerizable group independentlyselected from an acrylate group, a methacrylate group, an ethacrylategroup, a 2-phenyl acrylate group, an acrylamide group, a methacrylamidegroup, an itaconate group, and a styrene group.

134. The three-dimensional network of embodiment 133, wherein at leastone monomer species in the polymer comprises a methacrylate group.

135. The three-dimensional network of embodiment 134, wherein the atleast one monomer species comprising a methacrylate group ismethacryloyloxybenzophenone (MABP).

136. The three-dimensional network of embodiment 132, wherein thecrosslinked polymer comprises a polymer polymerized fromdimethylacrylamide (DMAA), methacryloyloxybenzophenone (MABP), andsodium 4-vinylbenzenesulfonate (SSNa).

137. The three-dimensional network of any one of embodiments 1 to 136,wherein the probe molecules are covalently attached to the network.

138. The three-dimensional network of any one of embodiments 1 to 137,wherein a majority of probe molecules are immobilized in the interior ofthe network.

139. The three-dimensional network of embodiment 138, wherein at least aportion of the probe molecules adjoin a channel.

140. In various embodiments, the probe density it at least 10%, at least20%, at least 30%, at least 40%, or at least 50% more dense at theinterface between the polymer and the channels than within regions ofthe polymer not adjacent to a channel.

141. The three-dimensional network of any one of embodiments 1 to 139,wherein the probe density at the interface between the polymer and thechannels is on average at least 10% greater than within regions of thepolymer not abutting a channel.

142. The three-dimensional network of embodiment 140, wherein the probedensity at the interface between the polymer and the channels is onaverage at least 20% greater than within regions of the polymer notabutting a channel.

143. The three-dimensional network of embodiment 140, wherein the probedensity at the interface between the polymer and the channels is onaverage at least 30% greater than within regions of the polymer notabutting a channel.

144. The three-dimensional network of embodiment 140, wherein the probedensity at the interface between the polymer and the channels is onaverage at least 40% greater than within regions of the polymer notabutting a channel.

145. The three-dimensional network of embodiment 140, wherein the probedensity at the interface between the polymer and the channels is onaverage at least 50% greater than within regions of the polymer notabutting a channel.

146. The three-dimensional network of any one of embodiments 1 to 145,wherein the probe molecules comprise a nucleic acid, a nucleic acidderivative, a peptide, a polypeptide, a protein, a carbohydrate, alipid, a cell, a ligand, or a combination thereof, preferably whereinthe probe molecules comprise a nucleic acid or a nucleic acidderivative.

147. The three-dimensional network of any one of embodiments 1 to 146,wherein the probe molecules comprise an antibody, an antibody fragment,an antigen, an epitope, an enzyme, an enzyme substrate, an enzymeinhibitor, a nucleic acid, or a combination thereof.

148. The three-dimensional network of any one of embodiments 1 to 147,wherein the probe molecules comprise a nucleic acid.

149. The three-dimensional network of embodiment 148, wherein thenucleic acid is an oligonucleotide.

150. The three-dimensional network of embodiment 149, wherein theoligonucleotide is 12 to 30 nucleotides long.

151. The three-dimensional network of embodiment 149, wherein theoligonucleotide is 14 to 30 nucleotides long.

152. The three-dimensional network of embodiment 149, wherein theoligonucleotide is 14 to 25 nucleotides long.

153. The three-dimensional network of embodiment 149, wherein theoligonucleotide is 14 to 20 nucleotides long.

154. The three-dimensional network of embodiment 149, wherein theoligonucleotide is 15 to 30 nucleotides long.

155. The three-dimensional network of embodiment 149, wherein theoligonucleotide is 15 to 25 nucleotides long.

156. The three-dimensional network of embodiment 149, wherein theoligonucleotide is 15 to 20 nucleotides long.

157. The three-dimensional network of embodiment 149, wherein theoligonucleotide is 16 to 30 nucleotides long.

158. The three-dimensional network of embodiment 149, wherein theoligonucleotide is 16 to 25 nucleotides long.

159. The three-dimensional network of embodiment 149, wherein theoligonucleotide is 16 to 20 nucleotides long.

160. The three-dimensional network of embodiment 149, wherein theoligonucleotide is 15 to 40 nucleotides long.

161. The three-dimensional network of embodiment 149, wherein theoligonucleotide is 15 to 45 nucleotides long.

162. The three-dimensional network of embodiment 149, wherein theoligonucleotide is 15 to 50 nucleotides long.

163. The three-dimensional network of embodiment 149, wherein theoligonucleotide is 15 to 60 nucleotides long.

164. The three-dimensional network of embodiment 149, wherein theoligonucleotide is 20 to 55 nucleotides long.

165. The three-dimensional network of embodiment 149, wherein theoligonucleotide is 18 to 60 nucleotides long.

166. The three-dimensional network of embodiment 149, wherein theoligonucleotide is 20 to 50 nucleotides long.

167. The three-dimensional network of embodiment 149, wherein theoligonucleotide is 30 to 90 nucleotides long.

168. The three-dimensional network of embodiment 149, wherein theoligonucleotide is 20 to 100 nucleotides long.

169. The three-dimensional network of embodiment 149, wherein theoligonucleotide is 20 to 60 nucleotides long.

170. The three-dimensional network of embodiment 149, wherein theoligonucleotide is 40 to 80 nucleotides long.

171. The three-dimensional network of embodiment 149, wherein theoligonucleotide is 40 to 100 nucleotides long.

172. The three-dimensional network of embodiment 149, wherein theoligonucleotide is 20 to 120 nucleotides long.

173. The three-dimensional network of embodiment 149, wherein theoligonucleotide is 20 to 40 nucleotides long.

174. The three-dimensional network of embodiment 149, wherein theoligonucleotide is 40 to 60 nucleotides long.

175. The three-dimensional network of embodiment 149, wherein theoligonucleotide is 60 to 80 nucleotides long.

176. The three-dimensional network of embodiment 149, wherein theoligonucleotide is 80 to 100 nucleotides long.

177. The three-dimensional network of embodiment 149, wherein theoligonucleotide is 100 to 120 nucleotides long.

178. The three-dimensional network of embodiment 149, wherein theoligonucleotide is 12 to 150 nucleotides long.

179. An array comprising a plurality of three-dimensional networksaccording to any one of embodiments 1 to 178 on a substrate.

180. The array of embodiment 179, wherein the three-dimensional networksare immobilized on the substrate.

181. The array of embodiment 180, wherein the three-dimensional networksare immobilized on the substrate by covalent bonds between the networksand the substrate.

182. The array of any one of embodiments 179 to 181, wherein thesubstrate comprises an organic polymer or an inorganic material having aself-assembled monolayer of organic molecules on a surface of theinorganic material.

183. The array of embodiment 182, wherein the substrate comprises anorganic polymer selected from cycloolefin copolymers, polystyrene,polyethylene, polypropylene and polymethylmethacrylate.

184. The array of embodiment 183, wherein the substrate comprises acycloolefin copolymer, polystyrene or polymethylmethacrylate.

185. The array of embodiment 182, wherein the substrate comprises aninorganic material having an alkyl silane self-assembled monolayer on asurface of the inorganic material.

186. The array of any one of embodiments 179 to 185 which comprises atleast 8 three-dimensional networks.

187. The array of any one of embodiments 179 to 185 which comprises atleast 16 three-dimensional networks.

188. The array of any one of embodiments 179 to 185 which comprises atleast 24 three-dimensional networks.

189. The array of any one of embodiments 179 to 185 which comprises atleast 48 three-dimensional networks.

190. The array of any one of embodiments 179 to 185 which comprises atleast 96 three-dimensional networks.

191. The array of any one of embodiments 179 to 185 which comprises atleast 128 three-dimensional networks.

192. The array of any one of embodiments 179 to 185 which comprises atleast 256 three-dimensional networks.

193. The array of any one of embodiments 179 to 185 which comprises atleast 512 three-dimensional networks.

194. The array of any one of embodiments 179 to 185 which comprises atleast 1024 three-dimensional networks.

195. The array of any one of embodiments 179 to 185 which comprises 24to 8192 three-dimensional networks.

196. The array of any one of embodiments 179 to 185 which comprises 24to 4096 three-dimensional networks.

197. The array of any one of embodiments 179 to 185 which comprises 24to 2048 three-dimensional networks.

198. The array of any one of embodiments 179 to 185 which comprises 24to 1024 three-dimensional networks.

199. The array of any one of embodiments 179 to 185 which comprises 24three-dimensional networks.

200. The array of any one of embodiments 179 to 185 which comprises 48three-dimensional networks.

201. The array of any one of embodiments 179 to 185 which comprises 96three-dimensional networks.

202. The array of any one of embodiments 179 to 185 which comprises 128three-dimensional networks.

203. The array of any one of embodiments 179 to 185 which comprises 256three-dimensional networks.

204. The array of any one of embodiments 179 to 185 which comprises 512three-dimensional networks.

205. The array of any one of embodiments 179 to 185 which comprises 1024three-dimensional networks.

206. The array of any one of embodiments 179 to 205, wherein each of thethree-dimensional networks is located at a separate spot on thesubstrate.

207. The array of embodiment 206, wherein the spots are arranged incolumns and/or rows.

208. The array of any one of embodiments 179 to 207, wherein thesubstrate comprises a microwell plate and the three-dimensional networksare positioned in the wells of the plate.

209. The array of any one of embodiments 179 to 208, wherein theplurality of three-dimensional networks comprises two or morethree-dimensional networks comprising different species of probemolecules.

210. The array of any one of embodiments 179 to 209, wherein theplurality of three-dimensional networks comprises two or morethree-dimensional networks comprising the same species of probemolecules.

211. The array of any one of embodiments 179 to 210, wherein a majorityof the three-dimensional networks comprise the same species of probemolecules or wherein all the three-dimensional networks comprises thesame species of probe molecules.

212. The array of any one of embodiments 179 to 211, wherein theplurality of three-dimensional networks comprises one or morethree-dimensional networks comprising labeled control probe molecules.

213. The array of embodiment 212, wherein the labeled control probemolecules are fluorescently labeled.

214. The array of embodiment 212 or embodiment 213, wherein at least onecontrol probe molecule is a spatial control.

215. The array of any one of embodiments 179 to 214, which can bereused.

216. The array of embodiment 215, which can be reused at least 5 times.

217. The array of embodiment 215, which can be reused at least 10 times.

218. The array of embodiment 215, which can be reused at least 20 times.

219. The array of embodiment 215, which can be reused at least 30 times

220. The array of embodiment 215, which can be reused at least 40 times.

221. The array of embodiment 215, which can be reused at least 50 times.

222. The array of embodiment 215, which can be reused at least 60 times.

223. The array of embodiment 215, which can be reused at least 70 times

224. The array of embodiment 215, which can be reused at least 80 times.

225. The array of embodiment 215, which can be reused at least 90 times.

226. The array of embodiment 215, which can be reused at least 100times.

227. The array of any one of embodiments 215 to 226, in which at leastone three-dimensional network is a reusability control.

228. The array of embodiment 227, wherein the reusability controlcomprises a fluorescently labeled probe.

229. The array of embodiment 228, wherein the reusability control isalso a spatial control.

230. A process for making a three-dimensional network having a surfaceand an interior comprising (a) a crosslinked polymer and (b) one or morechannels, comprising:

-   -   (a) exposing a mixture to salt crystal forming conditions, said        mixture comprising (i) an aqueous salt solution, (ii) a polymer,        and (iii) a cross-linker and optionally positioned on the        surface of a substrate, thereby forming a mixture containing one        or more salt crystals;    -   (b) exposing the mixture containing one or more salt crystals to        crosslinking conditions, thereby forming a crosslinked polymer        network containing one or more salt crystals; and    -   (c) contacting the crosslinked polymer network containing one or        more salt crystals with a solvent in which the one or more salt        crystals are soluble, thereby dissolving the salt crystals and        forming one or more channels in place of the salt crystals;

thereby forming the three-dimensional network comprising a crosslinkedpolymer and one or more channels.

231. The process of embodiment 230, wherein the salt forming conditionscomprise forming one or more needle-shaped crystals.

232. The process of embodiment 230 or embodiment 231, in which at leastone of the one or more channels has the properties of the channels ofthe networks of any one of embodiments 2 to 51.

233. The process of embodiment 230 or embodiment 231, in which athree-dimensional network having a surface and an interior comprising(a) a crosslinked polymer and (b) a plurality of channels is produced.

234. The process of embodiment 233, in which the plurality of channelshave the properties of the plurality of channels of the networks of anyone of embodiments 53 to 112.

235. The process of any one of embodiments 230 to 234, wherein the saltforming conditions comprise dehydrating the mixture.

236. The process of embodiment 235, which comprises dehydrating themixture by heating the mixture, exposing the mixture to a vacuum,reducing the humidity of the atmosphere surrounding the mixture, or acombination thereof.

237. The process of embodiment 236, which comprises dehydrating themixture by exposing the mixture to a vacuum.

238. The process of embodiment 236, which comprises dehydrating themixture by heating the mixture.

239. The process of embodiment 238, wherein heating the mixturecomprises contacting the mixture with a gas that has a temperature whichis higher than the temperature of the mixture.

240. The process of any one of embodiments 230 to 234, wherein the saltforming conditions comprise cooling the mixture until the mixturebecomes supersaturated with the salt.

241. The process of embodiment 240, which comprises cooling the mixtureby contacting the mixture with a gas that has a temperature which islower than the temperature of the mixture.

242. The process of any one of embodiments 230 to 241, wherein thetemperature of the mixture during step (a) is maintained above the dewpoint of the atmosphere surrounding the mixture.

243. The process of any one of embodiments 230 to 242, wherein thecross-linker is activated by ultraviolet (UV) light and the crosslinkingconditions comprise exposing the mixture to ultraviolet light.

244. The process of any one of embodiments 230 to 242, wherein thecross-linker is activated by visible light and the crosslinkingconditions comprise exposing the mixture to visible light.

245. The process of any one of embodiments 230 to 242, wherein thecross-linker is activated by heat and the crosslinking conditionscomprise exposing the mixture to heat.

246. The process of any one of embodiments 230 to 245, wherein theaqueous salt solution comprises monovalent cations.

247. The process of embodiment 246, in which the monovalent cationscomprise Na⁺ and/or K⁺, preferably wherein the monovalent cationscomprise Na⁺ and K⁺.

248. The process of embodiment 247, wherein the aqueous salt solutioncomprises a solution produced by a process comprising dissolvingdisodium hydrogen phosphate, sodium dihydrogen phosphate, dipotassiumhydrogen phosphate, potassium dihydrogen phosphate, or a combinationthereof in water or an aqueous solution.

249. The process of any one of embodiments 230 to 248, wherein thepolymer is a water-soluble polymer.

250. The process of any one of embodiments 230 to 249, wherein thepolymer comprises a homopolymer.

251. The process of any one of embodiments 230 to 249, wherein thepolymer comprises a copolymer.

252. The process of any one of embodiments 230 to 249, wherein thepolymer comprises a mixture of a homopolymer and a copolymer.

253. The process of any one of embodiments 249 to 252, wherein thepolymer comprises a polymer polymerized from one or more species ofmonomers.

254. The process of embodiment 253, wherein each species of monomercomprises a polymerizable group independently selected from an acrylategroup, a methacrylate group, an ethacrylate group, a 2-phenyl acrylategroup, an acrylamide group, a methacrylamide group, an itaconate group,and a styrene group.

255. The process of embodiment 254, wherein at least one monomer speciesin the polymer comprises a methacrylate group.

256. The process of embodiment 255, wherein the at least one monomerspecies comprising a methacrylate group is methacryloyloxybenzophenone(MABP).

257. The process of any one of embodiment 253, wherein the polymercomprises a polymer polymerized from dimethylacrylamide (DMAA),methacryloyloxybenzophenone (MABP), and sodium 4-vinylbenzenesulfonate(SSNa).

258. The process of any one of embodiments 230 to 257, wherein thepolymer is a copolymer comprising the cross-linker.

259. The process of embodiment 258, wherein the polymer comprises atleast two cross-linkers per polymer molecule.

260. The process of any one of embodiments 230 to 259, wherein thecross-linker is selected from benzophenone, a thioxanthone, a benzoinether, ethyl eosin, eosin Y, rose bengal, camphorquinone, erythrosin,4,4′ azobis(4-cyanopentanoic) acid, 2,2-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride, and benzoyl peroxide.

261. The process of claim 260, wherein the cross-linker is benzophenone.

262. The process of any one of embodiments 230 to 261, wherein thesolvent is water or a water-based buffer.

263. The process of embodiment 262, wherein the solvent is water.

264. The process of embodiment 262, wherein the solvent is a water-basedbuffer.

265. The process of embodiment 264, wherein the water-based buffercomprises phosphate, methanol, ethanol, propanol, or a mixture thereof.

266. The process of any one of embodiments 230 to 265, wherein themixture of step (a) further comprises probe molecules.

267. The process of embodiment 266, wherein at least some, the majorityor all the probe molecules comprise a nucleic acid, a nucleic acidderivative, a peptide, a polypeptide, a protein, a carbohydrate, alipid, a cell, a ligand, or a combination thereof.

268. The process of embodiment 267, wherein at least some of the probemolecules comprise a nucleic acid or a nucleic acid derivative.

269. The process of embodiment 267, wherein at least a majority of theprobe molecules comprise a nucleic acid or a nucleic acid derivative.

270. The process of embodiment 267, wherein all the probe moleculescomprise a nucleic acid or a nucleic acid derivative.

271. The process of embodiment 266, wherein at least some, the majorityor all the probe molecules comprise an antibody, an antibody fragment,an antigen, an epitope, an enzyme, an enzyme substrate, an enzymeinhibitor, a nucleic acid, or a combination thereof.

272. The process of embodiment 271, wherein at least some of the probemolecules comprise a nucleic acid.

273. The process of embodiment 271, wherein at least a majority of theprobe molecules comprise a nucleic acid.

274. The process of embodiment 271, wherein all the probe moleculescomprise a nucleic acid.

275. The process of any one of embodiments 272 to 274, wherein thenucleic acid is an oligonucleotide.

276. The process of embodiment 275, wherein the oligonucleotide is 12 to30 nucleotides long.

277. The process of embodiment 275, wherein the oligonucleotide is 14 to30 nucleotides long.

278. The process of embodiment 275, wherein the oligonucleotide is 14 to25 nucleotides long.

279. The process of embodiment 275, wherein the oligonucleotide is 14 to20 nucleotides long.

280. The process of embodiment 275, wherein the oligonucleotide is 15 to30 nucleotides long.

281. The process of embodiment 275, wherein the oligonucleotide is 15 to25 nucleotides long.

282. The process of embodiment 275, wherein the oligonucleotide is 15 to20 nucleotides long.

283. The process of embodiment 275, wherein the oligonucleotide is 16 to30 nucleotides long.

284. The process of embodiment 275, wherein the oligonucleotide is 16 to25 nucleotides long.

285. The process of embodiment 275, wherein the oligonucleotide is 16 to20 nucleotides long.

286. The process of embodiment 275, wherein the oligonucleotide is 15 to40 nucleotides long.

287. The process of embodiment 275, wherein the oligonucleotide is 15 to45 nucleotides long.

288. The process of embodiment 275, wherein the oligonucleotide is 15 to50 nucleotides long.

289. The process of embodiment 275, wherein the oligonucleotide is 15 to60 nucleotides long.

290. The process of embodiment 275, wherein the oligonucleotide is 20 to55 nucleotides long.

291. The process of embodiment 275, wherein the oligonucleotide is 18 to60 nucleotides long.

292. The process of embodiment 275, wherein the oligonucleotide is 20 to50 nucleotides long.

293. The process of embodiment 275, wherein the oligonucleotide is 30 to90 nucleotides long.

294. The process of embodiment 275, wherein the oligonucleotide is 20 to100 nucleotides long.

295. The process of embodiment 275, wherein the oligonucleotide is 20 to120 nucleotides long.

296. The process of embodiment 275, wherein the oligonucleotide is 20 to40 nucleotides long.

297. The process of embodiment 275, wherein the oligonucleotide is 20 to60 nucleotides long.

298. The process of embodiment 275, wherein the oligonucleotide is 40 to80 nucleotides long.

299. The process of embodiment 275, wherein the oligonucleotide is 40 to100 nucleotides long.

300. The process of embodiment 275, wherein the oligonucleotide is 40 to60 nucleotides long.

301. The process of embodiment 275, wherein the oligonucleotide is 60 to80 nucleotides long.

302. The process of embodiment 275, wherein the oligonucleotide is 80 to100 nucleotides long.

303. The process of embodiment 275, wherein the oligonucleotide is 100to 120 nucleotides long.

304. The process of embodiment 275, wherein the oligonucleotide is 12 to150 nucleotides long.

305. The process of any one of embodiments 230 to 304, furthercomprising, prior to step (a), a step of applying the mixture to asurface of a substrate.

306. The process of embodiment 305, wherein the mixture is applied in avolume of in a volume of 100 pl to 5 nl.

307. The process of embodiment 305, wherein the mixture is applied in avolume of in a volume of 100 pl to 1 nl.

308. The process of embodiment 305, wherein the mixture is applied in avolume of in a volume of 200 pl to 1 nl.

309. The process of any one of embodiments 305 to 308, wherein the stepof applying the mixture to the substrate comprises spraying the mixtureonto the surface of the substrate.

310. The process of embodiment 309, wherein the mixture is sprayed by aninkjet printer.

311. The process of any one of embodiments 305 to 310, wherein thesubstrate comprises an organic polymer or an inorganic material having aself-assembled monolayer of organic molecules on the surface.

312. The process of embodiment 311, wherein the substrate comprises anorganic polymer.

313. The process of embodiment 312, wherein the organic polymer isselected from cycloolefin copolymers, polystyrene, polyethylene,polypropylene and polymethylmethacrylate.

314. The process of embodiment 313, wherein the substrate comprisespolymethylmethacrylate, polystyrene, or cycloolefin copolymers.

315. The process of embodiment 311, wherein the substrate comprises aninorganic material having an alkyl silane self-assembled monolayer onthe surface.

316. The process of any one of embodiments 305 to 315, wherein thesubstrate comprises a microwell plate.

317. The process of any one of embodiments 305 to 316, wherein thepolymer is crosslinked to the surface in step (b).

318. The process of embodiment 317, in which a water-swellable polymeris produced that is crosslinked to the surface.

319. The process of embodiment 318, wherein the water-swellable polymercan absorb up to 50 times its weight of deionized, distilled water.

320. The process of embodiment 318 or embodiment 319, wherein thewater-swellable polymer can absorb 5 to 50 times its own volume ofdeionized, distilled water.

321. The process of any one of embodiments 318 to 320, wherein thewater-swellable polymer can absorb up to 30 times its weight of saline.

322. The process of any one of embodiments 318 to 321, wherein thewater-swellable polymer can absorb 4 to 30 times its own volume ofsaline.

323. A process for making an array, comprising generating a plurality ofthree-dimensional networks by the process of any one of claims 230 to322 at discrete spots on the surface of the same substrate.

324. The process of embodiment 323, wherein the three-dimensionalnetworks are generated simultaneously.

325. The process of embodiment 323, wherein the three-dimensionalnetworks are generated sequentially.

326. The process of any one of embodiments 323 to 325, furthercomprising crosslinking the plurality of three-dimensional networks tothe surface of the substrate.

327. A process for making an array, comprising positioning a pluralityof three-dimensional networks (a) according to any one of embodiments 1to 178 or (b) produced or obtainable according to the process of any oneof embodiments 230 to 322 at discrete spots on a surface of the samesubstrate.

328. The process of any one of embodiments 323 to 327, furthercomprising crosslinking the plurality of three-dimensional networks tothe surface.

329. A process for making an array, comprising positioning a pluralityof three-dimensional networks produced or obtainable according to theprocess of any one of embodiments 305 to 322 at discrete spots on asurface of the same substrate.

330. The process of embodiment 329, wherein the positioning comprisesapplying the mixtures from which the three-dimensional networks areformed at the discrete spots.

331. The process of any one of embodiments 323 to 330, wherein the spotsare arranged in columns and/or rows.

332. A three-dimensional network produced or obtainable by the processof any one of embodiments 230 to 322.

333. An array comprising a plurality of three-dimensional networksaccording to embodiment 332 on a substrate.

334. An array produced or obtainable by the process of any one ofembodiments 323 to 331.

335. The array of embodiment 333 or embodiment 334 which comprises atleast 8 three-dimensional networks.

336. The array of embodiment 333 or embodiment 334 which comprises atleast 16 three-dimensional networks.

337. The array of embodiment 333 or embodiment 334 which comprises atleast 24 three-dimensional networks.

338. The array of embodiment 333 or embodiment 334 which comprises atleast 48 three-dimensional networks.

339. The array of embodiment 333 or embodiment 334 which comprises atleast 96 three-dimensional networks.

340. The array of embodiment 333 or embodiment 334 which comprises atleast 128 three-dimensional networks.

341. The array of embodiment 333 or embodiment 334 which comprises atleast 256 three-dimensional networks.

342. The array of embodiment 333 or embodiment 334 which comprises atleast 512 three-dimensional networks.

343. The array of embodiment 333 or embodiment 334 which comprises atleast 1024 three-dimensional networks.

344. The array of embodiment 333 or embodiment 334 which comprises 24 to8192 three-dimensional networks.

345. The array of embodiment 333 or embodiment 334 which comprises 24 to4096 three-dimensional networks.

346. The array of embodiment 333 or embodiment 334 which comprises 24 to2048 three-dimensional networks.

347. The array of embodiment 333 or embodiment 334 which comprises 24 to1024 three-dimensional networks.

348. The array of embodiment 333 or embodiment 334 which comprises 24three-dimensional networks.

349. The array of embodiment 333 or embodiment 334 which comprises 48three-dimensional networks.

350. The array of embodiment 333 or embodiment 334 which comprises 96three-dimensional networks.

351. The array of embodiment 333 or embodiment 334 which comprises 128three-dimensional networks.

352. The array of embodiment 333 or embodiment 334 which comprises 256three-dimensional networks.

353. The array of embodiment 333 or embodiment 334 which comprises 512three-dimensional networks.

354. The array of embodiment 333 or embodiment 334 which comprises 1024three-dimensional networks.

355. The array of any one of embodiments 333 to 354, wherein thethree-dimensional networks comprise probe molecules, and two or more ofthree-dimensional networks comprise different species of probemolecules.

356. The array of any one of embodiments 333 to 355, wherein thethree-dimensional networks comprise probe molecules, and two or morethree-dimensional networks comprise the same species of probe molecules.

357. The array of any one of embodiments 333 to 354, wherein thethree-dimensional networks comprise probe molecules, and each of thethree-dimensional networks comprise the same species of probe molecules.

358. The array of any one of embodiments 333 to 357, wherein theplurality of three-dimensional networks comprises one or morethree-dimensional networks comprising labeled control probe molecules.

359. The array of embodiment 358, wherein the labeled control probemolecules are fluorescently labeled.

360. The array of any one of embodiments 333 to 359, wherein thesubstrate comprises a microwell plate and each well of the microwellplate contains no more than a single three-dimensional network.

361. A method for determining whether an analyte is present in a sample,comprising:

-   -   (a) contacting a three-dimensional network according to any one        of embodiments 1 to 178 or 332 or an array of any one of        embodiments 179 to 229 or 333 to 360 comprising probe molecules        that are capable of binding to the analyte with the sample; and    -   (b) detecting binding of the analyte to the probe molecules in        the three-dimensional network or array, thereby determining        whether the analyte is present in the sample.

362. The method of embodiment 361, which further comprises washing thenetwork or array comprising probe molecules between steps (a) and (b).

363. The method of embodiment 361 or embodiment 362, which furthercomprises contacting the network or array comprising probe moleculeswith a blocking reagent prior to step (a).

364. The method of any one of embodiments 361 to 363, further comprisingquantifying the amount of analyte bound to the three-dimensional networkor array comprising probe molecules.

365. A method for determining whether an analyte is present in eachsample in a plurality of samples, comprising:

-   -   (a) contacting an array of any one of embodiments 179 to 229 or        333 to 360 comprising probe molecules that are capable of        binding to the analyte with the samples; and    -   (b) detecting binding of the analyte to the probe molecules in        the array, thereby determining whether the analyte is present in        each sample in the plurality of samples.

366. A method for determining whether an analyte is present in eachsample in a plurality of samples, comprising:

-   -   (a) contacting an array of any one of embodiments 179 to 229 or        333 to 360 comprising probe molecules that are capable of        binding to the analyte with the samples and comprising control        probe molecules, wherein the array has been used and washed        prior to step (a); and    -   (b) detecting binding of the analyte to the probe molecules in        the array, thereby determining whether the analyte is present in        each sample in the plurality of samples.

367. A method for determining whether more than one species of analyteis present in a sample, comprising:

-   -   (a) contacting an array of any one of embodiments 179 to 229 or        333 to 360 comprising different species of probe molecules that        are capable of binding to the different species of analytes with        the sample; and    -   (b) detecting binding of the analytes to the probe molecules in        the array, thereby determining whether more than one species of        analyte are present in the sample.

368. A method for determining whether more than one species of analyteis present in a sample, comprising:

-   -   (a) contacting an array of any one of embodiments 179 to 229 or        333 to 360 comprising different species of probe molecules that        are capable of binding to the different species of analytes with        the sample and comprising control probe molecules, wherein the        array has been used and washed prior to step (a); and    -   (b) detecting binding of the analytes to the probe molecules in        the array, thereby determining whether more than one species of        analyte are present in the sample.

369. The method of any one of embodiments 365 to 368, in which:

-   -   (a) the substrate of the array comprises a microwell plate;    -   (b) each well of the microwell plate contains no more than a        single three-dimensional network; and    -   (c) contacting the array with the samples comprises contacting        each well with no more than a single sample.

370. The method of any one of embodiments 365 to 369, which furthercomprises washing the array comprising probe molecules between steps (a)and (b).

371. The method of any one of embodiments 365 to 370, which furthercomprises contacting the array comprising probe molecules with ablocking reagent prior to step (a).

372. The method of any one of embodiments 365 to 371, further comprisingquantifying the amount of analyte or analytes bound to the array.

373. The method of any one of embodiments 361 to 372, further comprisingreusing the array.

374. The method of embodiment 373, wherein the array is reused at least5 times.

375. The method of embodiment 373, wherein the array is reused at least10 times.

376. The method of embodiment 373, wherein the array is reused at least20 times.

377. The method of embodiment 373, wherein the array is reused at least30 times.

378. The method of embodiment 373, wherein the array is reused at least40 times.

379. The method of embodiment 373, wherein the array is reused at least50 times.

380. The method of embodiment 374, which comprises reusing the array 5to 20 times.

381. The method of embodiment 374, which comprises reusing the array 5to 30 times.

382. The method of embodiment 374, which comprises reusing the array 10to 50 times.

383. The method of embodiment 374, which comprises reusing the array 10to 20 times.

384. The method of embodiment 374, which comprises reusing the array 10to 30 times.

385. The method of embodiment 374, which comprises reusing the array 20to 40 times.

386. The method of embodiment 374, which comprises reusing the array 40to 50 times.

387. The method of any one of embodiments 373 to 386, which compriseswashing the array between reuses.

388. The method of embodiment 387, wherein the array is washed underdenaturing conditions.

389. The method of embodiment 388 wherein the denaturing conditionscomprise exposing the array to heat.

390. The method of embodiment 388 wherein the denaturing conditionscomprise exposing the array to low salt concentrations.

391. The method of embodiment 388 wherein the denaturing conditionscomprise exposing the array to both heat and low salt concentrations.

392. The method of embodiment 388, wherein the denaturing conditions areremoved prior to reuse.

393. The method of embodiment 392, wherein the denaturing conditionscomprise exposing the array to heat and wherein the temperature islowered prior to reuse.

394. The method of embodiment 392, wherein the denaturing conditionscomprise exposing the array to low salt concentrations and wherein thesalt concentration is increased prior to reuse.

395. The method of embodiment 392, wherein the denaturing conditionscomprise exposing the array to both heat and low salt concentrations andwherein the temperature is lowered and the salt concentration isincreased prior to reuse.

396. The method of any one of embodiments 373 to 395, wherein the arraycomprises at least one three-dimensional network comprising afluorescently labelled oligonucleotide as a reusability control.

397. The method of embodiment 396, which comprises testing thefluorescent signal strength.

398. The method of embodiment 397, wherein the reusability controlretains at least 70% of its initial fluorescence signal strength after10 uses.

399. The method of embodiment 398, wherein the reusability controlretains at least 50% of its signal strength after 20 uses.

400. The method of any one of embodiments 396 to 399, wherein the arrayis no longer reused after the reusability control loses more than 50% ofits signal strength.

401. The method of any one of embodiments 361 to 400, wherein analyte isa nucleic acid.

402. The method of embodiment 401, wherein the nucleic acid is apolymerase chain reaction (PCR) amplicon.

403. The method of embodiment 401, wherein the PCR amplicon is amplifiedfrom a biological sample or environmental sample.

404. The method of embodiment 403, wherein the PCR amplicon is amplifiedfrom a biological sample.

405. The method of embodiment 403, wherein the PCR amplicon is amplifiedfrom an environmental sample.

406. The method of embodiment 404, wherein the biological sample is ablood, serum, plasma, tissue, cells, saliva, sputum, urine,cerebrospinal fluid, pleural fluid, milk, tears, stool, sweat, semen,whole cells, cell constituent, cell smear, or an extract or derivativethereof.

407. The method of embodiment 406, wherein the biological sample ismammalian blood, serum or plasma or an extract thereof.

408. The method of embodiment 407, wherein the biological sample ishuman or bovine blood, serum or plasma or an extract thereof.

409. The method of embodiment 406, wherein the biological sample is milkor an extract thereof.

410. The method of embodiment 409, wherein the biological sample iscow's milk or an extract thereof.

411. The method of any one of embodiments 401 to 410, wherein nucleicacid is labeled.

412. The method of embodiment 411, wherein the nucleic acid isfluorescently labeled.

413. A three-dimensional network (15) having a surface (16) and aninterior comprising:

-   -   (a) a crosslinked polymer (3) covalently attached to the surface        (2) of a substrate;    -   (b) one or more channels (13); and    -   (c) probe molecules immobilized (1) in the network (15),        optionally

wherein

-   -   (i) probe molecules (1) are covalently attached to the network        (15) and/or    -   (ii) a majority of probe molecules (1) are immobilized in the        interior of the network (15) and/or    -   (iii) a majority of probe molecules (1) adjoin a channel (13).

414. The three-dimensional network (15) of embodiment 413, wherein atleast one or at least a majority of the channels (13) is characterizedby one, two, or three of the following properties:

-   -   (a) the channel (13) extends into the interior from a point that        is less than 5 microns from the surface (16) of the network (15)        or extends into the interior from a point on the surface (16) of        the network (15);    -   (b) the channel (13) has a length that is at least 10 or at        least 20% of the largest dimension of the network (15); and    -   (c) the channel (13) has a minimum cross-section of at least 5        times or at least 15 times the network's (15) mesh size.

415. The three-dimensional network (15) of embodiment 414, wherein atleast one or at least a majority of the channels (13)

-   -   (a) has a length that is 10% to 40% or 15% to 25% of the largest        dimension of the network (15) and/or    -   (b) has a minimum cross-section of 5 to 10 times or 10 to 25        times the network's (15) mesh size.

416. The three-dimensional network (15) of any one of embodiments 413 to415, comprising at least 5 channels (13), at least 10 channels (13) orat least 15 channels (13), optionally wherein a plurality of channels(13) converge at a point in the interior of the network (15) such thatthe lateral distance between the channels (13) decreases from thesurface (16) of the network (15) toward the point in the interior, andoptionally wherein each channel (13) is independently characterized byone, two, or three of the following properties:

-   -   (a) the channel (13) extends into the interior from a point that        is less than 10 microns, less than 9 microns, less than 8        microns, less than 7 microns, less than 6 microns, less than 5        microns, less than 4 microns, less than 3 microns, less than 2        microns, less than 1 micron from the surface (16) of the network        (15) or on the surface (16) of the network (15);    -   (b) the channel (13) has a length that is at least 10%, at least        15%, at least 20%, or at least 25% of the largest dimension of        the network (15), and; and/or    -   (c) the channel (13) has a minimum cross-section of at least 5        times, at least 10 times, at least 15 times, or at least 20        times the network's (15) mesh size.

417. The three-dimensional network (15) of any one of embodiments 413 to416, wherein the network (15) has in its hydrated state a mesh size of 5to 75 nm or 10 to 50 nm.

418. An array comprising a plurality of three-dimensional networks (15)according to any one of embodiments 413 to 417 on a substrate,optionally wherein (a) the three-dimensional networks (15) areimmobilized on the substrate and/or (b) each of the three-dimensionalnetworks (15) is located at a separate spot (7) on the substrate.

419. The array of embodiment 418, comprising at least 8 or at least 48three-dimensional networks (15), optionally wherein the number ofthree-dimensional networks (15) on the array ranges between 24 and 1024.

420. The array of embodiment 418 or embodiment 419, wherein theplurality of three-dimensional networks (15) comprises one or morethree-dimensional networks (15) comprising labeled control probemolecules (1), optionally wherein the labeled control probe molecules(1) are fluorescently labeled.

421. The array of any one of embodiments 418 to 420 which can be reused,optionally wherein the array can be reused at least 10 times.

422. A process for making a three-dimensional network (15) according toany one of embodiments 413 to 417, comprising the steps of:

-   -   (a) exposing a mixture (5) positioned on the surface (2) of a        substrate to needle-shaped crystal forming conditions, said        mixture (5) comprising        -   (i) an aqueous salt solution which is optionally a            monovalent cation salt solution,        -   (ii) a polymer, and (iii) a cross-linker, thereby forming a            mixture (5) containing one or more needle-shaped salt            crystals (8);    -   (b) exposing the mixture (5) containing one or more salt        crystals (8) to crosslinking conditions, thereby forming a        crosslinked polymer network (15) containing one or more        needle-shaped salt crystals (8); and    -   (c) contacting the crosslinked polymer network (15) containing        one or more salt crystals (8) with a solvent in which the one or        more salt crystals (8) are soluble, thereby dissolving the        needle-shaped salt crystals (8) and forming one or more channels        (13) in place of the salt crystals (8).

423. The process of embodiment 422, wherein:

-   -   (a) the needle-shaped crystal salt forming conditions comprise:        -   (i) dehydrating the mixture (5) optionally by heating the            mixture (5) (optionally by contacting the mixture (5) with a            gas that has a temperature which is higher than the            temperature of the mixture (5)), exposing the mixture (5) to            a vacuum, and/or reducing the humidity of the atmosphere            surrounding the mixture (5); or        -   (ii) cooling the mixture (5), optionally by contacting the            mixture (5) with a gas that has a temperature which is lower            than the temperature of the mixture (5); and/or    -   (b) wherein the solvent is a water-based buffer, said buffer        optionally comprising phosphate, methanol, ethanol, propanol, or        a mixture thereof.

424. The process of embodiment 422 or embodiment 423, wherein themixture (5) of step (a) further comprises probe molecules (1).

425. The process of any one of embodiments 422 to 424, furthercomprising, prior to step (a), a step of applying the mixture (5) to asurface (2) of a substrate, optionally in a volume of 100 μl to 5 nl, ina volume of 100 μl to 1 nl or in a volume 500 μl to 2 nl.

426. A process for making an array, comprising (a) creating a pluralityof three-dimensional networks (15) by the process of any one ofembodiments 422 to 425 at discrete spots (7) on the surface (2) of thesame substrate, and (b) crosslinking the plurality of three-dimensionalnetworks (15) to the surface (2) of the substrate.

427. A method for determining whether an analyte is present in a sample,comprising:

-   -   (a) contacting a three-dimensional network (15) according to any        one of embodiments 413 to 417 comprising probe (1) molecules        that are capable of binding to the analyte with the sample,        optionally wherein the three-dimensional network (15) is        positioned on an array according to any one of embodiments 418        to 421; and    -   (b) detecting and optionally quantifying binding of the analyte        to the probe molecules (1) in the three-dimensional network (15)        or array, thereby determining whether the analyte is present in        the sample and optionally the amount of the analyte.

428. The method of embodiment 427, wherein:

-   -   (a) the network (15) or array has been used and washed prior to        step (a), optionally at least 10 times, at least 20 times or at        least 50 times; and/or    -   (b) further comprising reusing the network (15) or array        following step (b), optionally at least 10 times, at least 20        times or at least 50 times.

429. The method of embodiment 427 or embodiment 428, wherein the analyteis a nucleic acid, optionally wherein the nucleic acid is afluorescently labeled polymerase chain reaction (PCR) amplicon.

While various specific embodiments have been illustrated and described,it will be appreciated that various changes can be made withoutdeparting from the spirit and scope of the disclosure(s).

10. CITATION OF REFERENCES

All publications, patents, patent applications and other documents citedin this application are hereby incorporated by reference in theirentireties for all purposes to the same extent as if each individualpublication, patent, patent application or other document wereindividually indicated to be incorporated by reference for all purposes.In the event that there is an inconsistency between the teachings of oneor more of the references incorporated herein and the presentdisclosure, the teachings of the present specification are intended.

The invention claimed is:
 1. A three-dimensional network having asurface and an interior, said three-dimensional network: (a) is composedof a water-swellable polymer formed by crosslinking water-solublepolymer chains; (b) is crosslinked to the surface of a substrate; (c)comprises a plurality of channels that converge at a point in theinterior of the network such that the lateral distance between thechannels decreases from the surface of the network toward the point inthe interior; and (d) comprises probe molecules covalently attached tothe polymer chains, wherein a majority of the probe molecules areimmobilized in the interior of the network and at least a portion of theprobe molecules adjoin a channel.
 2. The three-dimensional network ofclaim 1, wherein a majority of probe molecules adjoin a channel.
 3. Thethree-dimensional network of claim 1, wherein the polymer comprises apolymer polymerized from dimethylacrylamide (DMAA),methacryloyloxybenzophenone (MABP), and sodium 4-vinylbenzenesulfonate(SSNa).
 4. The three-dimensional network of claim 1, wherein the probemolecules comprise nucleic acids.
 5. The three-dimensional network ofclaim 4, wherein the nucleic acids are oligonucleotides.
 6. An arraycomprising a plurality of three-dimensional networks according to claim1, wherein each of the three-dimensional networks is located at aseparate spot on the substrate.
 7. The array of claim 6, which can bereused at least 10 times.
 8. The array of claim 6, wherein the pluralityof three-dimensional networks comprises two or more three-dimensionalnetworks comprising different species of probe molecules.
 9. The arrayof claim 6, wherein the plurality of three-dimensional networkscomprises one or more three-dimensional networks comprising labeledcontrol probe molecules.
 10. The array of claim 9, wherein the labeledcontrol probe molecules are fluorescently labeled.
 11. The array ofclaim 9, wherein at least one control probe molecule is a spatialcontrol probe.
 12. The array of claim 9, wherein at least one controlprobe molecule is a reusability control probe.
 13. The array of claim12, wherein the reusability control probe is also a spatial controlprobe.
 14. A process for making a three-dimensional network according toclaim 1, comprising the steps of: (a) exposing a mixture positioned onthe surface of the substrate to needle-shaped crystal formingconditions, said mixture comprising (i) an aqueous salt solution whichis a monovalent cation salt solution, (ii) a water-soluble polymer,(iii) a cross-linker, and (iv) probe molecules, thereby forming amixture containing needle-shaped salt crystals; (b) exposing the mixturecontaining the needle-shaped salt crystals to crosslinking conditions,thereby forming a crosslinked polymer network having probe moleculescrosslinked thereto and containing needle-shaped salt crystals; and (c)contacting the crosslinked polymer network containing the needle-shapedsalt crystals with a solvent in which the needle-shaped salt crystalsare soluble, thereby dissolving the needle-shaped salt crystals andforming channels in place of the needle-shaped salt crystals.
 15. Theprocess of claim 14, wherein: (a) the solvent is a water-based buffer;and/or; (b) the needle-shaped crystal salt forming conditions comprise:(i) dehydrating the mixture; or (ii) cooling the mixture.
 16. A processfor making an array, comprising (a) creating a plurality ofthree-dimensional networks by the process of claim 14 at discrete spotson the surface of the substrate, and (b) crosslinking the plurality ofthree-dimensional networks to the surface of the substrate.
 17. A methodfor determining whether an analyte is present in a sample, comprising:(a) contacting a three-dimensional network according to claim 1comprising probe molecules that are capable of binding to the analytewith the sample; and (b) detecting binding of the analyte to the probemolecules in the three-dimensional network, thereby determining whetherthe analyte is present in the sample and optionally the amount of theanalyte.
 18. The method of claim 17, wherein the three-dimensionalnetwork has been used and washed at least 10 times prior to (a) or whichfurther comprises reusing the three-dimensional network at least 10times following step (b).
 19. The method of claim 18, which furthercomprises quantifying binding of the analyte to the probe molecules inthe three-dimensional network.